Vaginal inserted estradiol pharmaceutical compositions and methods

ABSTRACT

According to various embodiments of this disclosure, pharmaceutical compositions comprising solubilized estradiol are provided. In various embodiments, such compositions are encapsulated in soft capsules which may be vaginally inserted for the treatment of vulvovaginal atrophy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to the following U.S. PatentApplications: U.S. Provisional Application Ser. No. 61/932,140, entitled“VAGINAL INSERTED ESTRADIOL PHARMACEUTICAL COMPOSITIONS AND METHODS”,which was filed on Jan. 27, 2014; U.S. Provisional Application Ser. No.61/894,411, entitled “SOLUBLE ESTRADIOL CAPSULE FOR VAGINAL INSERTION,”which was filed on Oct. 22, 2013; U.S. Provisional Application Ser. No.61/745,313, entitled “SOLUBLE ESTRADIOL CAPSULE FOR VAGINAL INSERTION,”which was filed on Dec. 21, 2012; U.S. Provisional Application Ser. No.61/889,483, entitled “Natural Combination Hormone ReplacementPharmaceutical compositions and Therapies,” which was filed on Oct. 10,2013; U.S. Provisional Application Ser. No. 61/661,302, entitled“ESTRADIOL PHARMACEUTICAL COMPOSITIONS,” which was filed on Jun. 18,2012; U.S. Provisional Application Ser. No. 61/662,265, entitled“PROGESTERONE PHARMACEUTICAL COMPOSITIONS,” which was filed on Jun. 20,2012; U.S. patent application Ser. No. 13/684,002, entitled “NaturalCombination Hormone Replacement Pharmaceutical compositions andTherapies,” which was filed Nov. 21, 2012; U.S. Patent ApplicationSerial No. PCT/US2013/023309, entitled “Transdermal Hormone ReplacementTherapies,” which was filed Jan. 25, 2013; and U.S. patent applicationSer. No. 13/843,362, entitled “Transdermal Hormone ReplacementTherapies,” which was filed Mar. 15, 2013. All aforementionedapplications are hereby incorporated by reference herein in theirentirety.

BACKGROUND

This application is directed to pharmaceutical compositions, methods,and devices related to hormone replacement therapy.

Postmenopausal women frequently suffer from atrophic vaginitis or vulvarand vaginal atrophy (hereinafter “vulvovaginal atrophy” or “VVA”) withsymptoms including, for example, vaginal dryness, vaginal odor, vaginalor vulvar irritation or itching, dysuria (pain, burning, or stingingwhen urinating), dysparuenia (vaginal pain associated with sexualactivity), or vaginal bleeding associated with sexual activity. Othersymptoms include soreness; with urinary frequency and urgency; urinarydiscomfort and incontinence also occurring (“estrogen-deficient urinarystate(s)”). One symptom of vaginal atrophy is an increased vaginal pH,which creates an environment more susceptible to infections. The mucosalepithelium of the VVA patients also reported to show signs of severeatrophy and upon cytological examination accompanied by an increasednumber of the parabasal cells and a reduced number of superficial cells.

Each of these VVA-related states manifest symptoms associated withdecreased estrogenization of the vulvovaginal tissue, and can even occurin women treated with oral administration of an estrogen-basedpharmaceutical drug product. Although VVA is most common with menopausalwomen, it can occur at any time in a woman's life cycle.

Estrogen treatment has proven to be very successful in controllingmenopausal symptoms, including vaginal atrophy (VVA). Several studieshave shown that the symptoms connected with vaginal atrophy are oftenrelieved by estrogen treatment given either systemically or topically.The existing treatments have numerous problems, for example complianceissues with patients not completing or continuing treatment due to theproblems associated with the form of treatment.

Accordingly, disclosed herein is, among other things, a new soft gelvaginal pharmaceutical composition and dosage form containingsolubilized estradiol for the treatment of VVA. The soft gel vaginalpharmaceutical composition has been designed to mitigate commonlimitations found with other vaginal forms of estradiol. The soft gelvaginal pharmaceutical composition is expected to ease vaginaladministration, provide improved safety of insertion, minimize vaginaldischarge following administration, and provide a more effective dosageform with improved efficacy, safety and patient compliance.

SUMMARY

According to various aspects and embodiments of this disclosure, a softgel vaginal pharmaceutical composition as a potential treatment forpost-menopausal women suffering with moderate to severe symptoms of VVAis provided.

Provided herein is a pessary comprising: a) a therapeutically effectiveamount of estradiol; and b) a solubilizing agent comprising a mediumchain oil.

In some embodiments, the pessary comprises about 1 μg to about 25 μg ofestradiol. For example, the pessary can include about 1 μg to about 10μg of estradiol; and about 10 μg to about 25 μg of estradiol.

In some embodiments, the estradiol is solubilized.

In some embodiments, the medium chain oil comprises at least one C6-C12fatty acid or a glycol, monoglyceride, diglyceride, or triglycerideester thereof.

In some embodiments, the solubilizing agent comprises at least one esterselected from the group consisting of: an ester of caproic fatty acid,an ester of caprylic fatty acid, an ester of capric fatty acid, andcombinations thereof. For example, the solubilizing agent can include acaprylic/capric triglyceride.

In some embodiments, the pessary further comprises a capsule. Forexample, the capsule can be a soft gelatin capsule.

Also provided herein is a pessary comprising: a) a therapeuticallyeffective amount of estradiol; b) a caprylic/capric triglyceride; c) anon-ionic surfactant comprising PEG-6 palmitostearate and ethyleneglycol palmitostearate; and d) a soft gelatin capsule.

In some embodiments, a pessary provided herein comprises about 25 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estradiol of about 19 pg*hr/ml toabout 29 pg*hr/ml; and 2) a corrected geometric mean area under thecurve (AUC)₀₋₂₄ of estradiol of about 75 pg*hr/ml to about 112 pg*hr/ml.

In some embodiments, a pessary provided herein comprises about 25 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone of about 9 pg*hr/ml to about14 pg*hr/ml; and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone of about 43 pg*hr/ml to about 65 pg*hr/ml.

In some embodiments, a pessary provided herein comprises about 25 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone sulfate of about 416 pg*hr/mlto about 613 pg*hr/ml; and 2) a corrected geometric mean area under thecurve (AUC)₀₋₂₄ of estrone sulfate of about 3598 pg*hr/ml to about 5291pg*hr/ml.

In some embodiments, a pessary provided herein comprises about to g ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estradiol of about 12 pg*hr/ml toabout 18 pg*hr/ml; and 2) a corrected geometric mean area under thecurve (AUC)₀₋₂₄ of estradiol of about 42 pg*hr/ml to about 63 pg*hr/ml.In some embodiments, the pessary further provides a corrected geometricmean time to peak plasma concentration (T_(max)) of estradiol of about 1hrs to about 3 hrs.

In some embodiments, a pessary provided herein comprises about 10 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone of about 4 pg*hr/ml to about 7pg*hr/ml; and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone of about 20 pg*hr/ml to about 31 pg*hr/ml. In someembodiments, the pessary further provides a corrected geometric meantime to peak plasma concentration (T_(max)) of estrone of about 4 hrs toabout 8 hrs.

In some embodiments, a pessary provided herein comprises about 10 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone sulfate of about 10 pg*hr/mlto about 16 pg*hr/ml; and 2) a corrected geometric mean area under thecurve (AUC)₀₋₂₄ of estrone sulfate of about 56 pg*hr/ml to about 84pg*hr/ml. In some embodiments, the pessary further provides a correctedgeometric mean time to peak plasma concentration (T_(max)) of estronesulfate of about 4 hrs to about 7 hrs.

In some embodiments, a pessary provided herein comprises about 4 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estradiol of about 4 pg*hr/ml to about8 pg*hr/ml; and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estradiol of about 16 pg*hr/ml to about 26 pg*hr/ml. Insome embodiments, the pessary further provides a corrected geometricmean time to peak plasma concentration (T_(max)) of estradiol of about0.25 hrs to about 2 hrs.

In some embodiments, a pessary provided herein comprises about 4 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone of about 1 pg*hr/ml to about 3pg*hr/ml; and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone of about 8 pg*hr/ml to about 13 pg*hr/ml. In someembodiments, the pessary further provides a corrected geometric meantime to peak plasma concentration (T_(max)) of estrone of about 1 hrs toabout 4 hrs.

In some embodiments, a pessary provided herein comprises about 4 μg ofestradiol, wherein administration of the pessary to a patient provides,in a plasma sample from the patient: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone sulfate of about 4 pg*hr/ml toabout 7 pg*hr/ml; and 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone sulfate of about 22 pg*hr/ml to about 34 pg*hr/ml.In some embodiments, the pessary further provides a corrected geometricmean time to peak plasma concentration (T_(max)) of estrone sulfate ofabout 1 hrs to about 3 hrs.

Also provided herein is a pessary comprising about 1 μg to about 25 μgof estradiol, wherein administration of the pessary to a patientprovides a corrected geometric mean peak plasma concentration (C_(max))of estradiol that is less than about 30 pg*hr/ml. For example,administration of the pessary to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estradiol that isless than about 18 pg*hr/ml.

In some embodiments, a pessary comprising about 1 μg to about 25 μg ofestradiol is provided, wherein administration of the pessary to apatient provides a corrected geometric mean area under the curve(AUC)0-24 of estradiol that is less than about 112 pg*hr/ml. Forexample, administration of the pessary to a patient provides a correctedgeometric mean area under the curve (AUC)0-24 of estradiol that is lessthan about 63 pg*hr/ml.

In some embodiments, a pessary comprising about 1 μg to about 25 μg ofestradiol is provided, wherein administration of the pessary to apatient provides a corrected geometric mean peak plasma concentration(C_(max)) of estrone that is less than about 14 pg*hr/ml. For example,administration of the pessary to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estrone that isless than about 7 pg*hr/ml.

In some embodiments, a pessary comprising about 1 μg to about 25 μg ofestradiol is provided, wherein administration of the pessary to apatient provides a corrected geometric mean area under the curve(AUC)0-24 of estrone that is less than about 65 pg*hr/ml. For example,administration of the pessary to a patient provides a correctedgeometric mean area under the curve (AUC)0-24 of estrone that is lessthan about 31 pg*hr/ml.

In some embodiments, a pessary comprising about 1 μg to about 25 μg ofestradiol is provided, wherein administration of the pessary to apatient provides a corrected geometric mean peak plasma concentration(C_(max)) of estrone sulfate that is less than about 613 pg*hr/ml. Forexample, administration of the pessary to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estrone sulfatethat is less than about 16 pg*hr/ml.

In some embodiments, a pessary comprising about 1 μg to about 25 μg ofestradiol is provided, wherein administration of the pessary to apatient provides a corrected geometric mean area under the curve(AUC)0-24 of estrone sulfate that is less than about 5291 pg*hr/ml. Forexample, administration of the pessary to a patient provides a correctedgeometric mean area under the curve (AUC)0-24 of estrone sulfate that isless than about 84 pg*hr/ml.

Further provided herein is a pessary comprising about 1 μg to about 25μg of estradiol, wherein administration of the pessary to the proximalregion of the vagina of a patient provides a therapeutically effectiveconcentration of estradiol over 24 hours in the proximal region of thevagina.

This disclosure also provides a method of treating an estrogen-deficientstate, the method comprising administering to a patient in need thereof,a pessary as provided herein. In some embodiments, a method of treatingvulvovaginal atrophy is provided, the method comprising administering toa patient in need thereof, a pessary as provided herein.

In some embodiments of the methods provided herein, treatment comprisesreducing the severity of one or more symptoms selected from the groupconsisting of: vaginal dryness, dyspareunia, vaginal or vulvarirritation, vaginal or vulvar burning, vaginal or vulvar itching,dysuria, and vaginal bleeding associated with sexual activity.

In some embodiments of the methods provided herein treatment comprisesreducing the vaginal pH of the patient. For example, treatment comprisesreducing the vaginal pH of the patient to a pH of less than about 5.0.

In some embodiments of the methods provided herein treatment comprises achange in cell composition of the patient. For example, the change incell composition comprises reducing the number of parabasal vaginalcells or increasing the number of superficial vaginal cells. In someembodiments, the number of parabasal vaginal cells in the patient arereduced by at least about 35% (e.g., at least about 50%). In someembodiments, the number of superficial vaginal cells are increased by atleast about 5% (e.g., at least about 35%).

Further provided herein is a method for reducing vaginal dischargefollowing administration of a pessary, the method comprisingadministering to a patient in need thereof, a pessary provided herein,wherein the vaginal discharge following administration of the pessary iscompared to the vaginal discharge following administration of areference drug.

DRAWINGS

The above-mentioned features and objects of the this disclosure willbecome more apparent with reference to the following description takenin conjunction with the accompanying drawings wherein like referencenumerals denote like elements and in which:

FIG. 1 is a flow diagram illustrating a process in accordance withvarious embodiments of the invention;

FIG. 2 illustrates a suppository in accordance with various embodimentsof the invention;

FIG. 3 is a linear plot of mean plasma estradiol—baseline adjustedconcentrations versus time (N=36);

FIG. 4 is a semi-logarithmic plot of mean plasma estradiol—baselineadjusted concentrations versus time (N=36);

FIG. 5 is a linear plot of mean plasma estrone—baseline adjustedconcentrations versus time (N=36);

FIG. 6 is a semi-logarithmic plot of mean plasma estrone—baselineadjusted concentrations versus time (N=36);

FIG. 7 is a linear plot of mean plasma estrone sulfate—baseline adjustedconcentrations versus time (N=36);

FIG. 8 is a semi-logarithmic plot of mean plasma estronesulfate—baseline adjusted concentrations versus time (N=36);

FIG. 9 is a linear plot of mean plasma estradiol—baseline adjustedconcentrations versus time (N=34);

FIG. 10 is a semi-logarithmic plot of mean plasma estradiol—baselineadjusted concentrations versus time (N=34);

FIG. 11 is a linear plot of mean plasma estrone—baseline adjustedconcentrations versus time (N=33);

FIG. 12 is a semi-logarithmic plot of mean plasma estrone—baselineadjusted concentrations versus time (N=33);

FIG. 13 is a linear plot of mean plasma estrone sulfate—baselineadjusted concentrations versus time (N=24); and

FIG. 14 is a semi-logarithmic plot of mean plasma estronesulfate—baseline adjusted concentrations versus time (N=24).

DETAILED DESCRIPTION

In the following detailed description of embodiments of this disclosure,reference is made to the accompanying drawings in which like referencesindicate similar elements, and in which is shown by way of illustrationspecific embodiments in which the this disclosure may be practiced.These embodiments are described in sufficient detail to enable thoseskilled in the art to practice the this disclosure, and it is to beunderstood that other embodiments may be utilized and that other changesmay be made without departing from the scope of the this disclosure. Thefollowing detailed description is, therefore, not to be taken in alimiting sense, and the scope of this disclosure is defined only by theappended claims. As used in this disclosure, the term “or” shall beunderstood to be defined as a logical disjunction (i.e., and/or) andshall not indicate an exclusive disjunction unless expressly indicatedas such with the terms “either,” “unless,” “alternatively,” and words ofsimilar effect.

Definitions

The term “active pharmaceutical ingredient” (“API”) as used herein,means the active compound(s) used in formulating a drug product.

The term “co-administered” as used herein, means that two or more drugproducts are administered simultaneously or sequentially on the same ordifferent days.

The term “drug product” as used herein means at least one activepharmaceutical ingredient in combination with at least one excipient andprovided in unit dosage form.

The term “area under the curve” (“AUC”) refers to the area under thecurve defined by changes in the blood concentration of an activepharmaceutical ingredient (e.g., estradiol or progesterone), or ametabolite of the active pharmaceutical ingredient, over time followingthe administration of a dose of the active pharmaceutical ingredient.“AUC_(0-∞)” is the area under the concentration-time curve extrapolatedto infinity following the administration of a dose. “AUC_(0-t)” is thearea under the concentration-time curve from time zero to time tfollowing the administration of a dose, wherein t is the last time pointwith a measurable concentration.

The term “C_(max)” refers to the maximum value of blood concentrationshown on the curve that represents changes in blood concentrations of anactive pharmaceutical ingredient (e.g., progesterone or estradiol), or ametabolite of the active pharmaceutical ingredient, over time.

The term “T_(max)” refers to the time that it takes for the bloodconcentration an active pharmaceutical ingredient (e.g., estradiol orprogesterone), or a metabolite of the active pharmaceutical ingredient,to reach the maximum value.

The term “bioavailability,” which has the meaning defined in 21 C.F.R. §320.1(a), refers to the rate and extent to which an API or activeingredient or active moiety is absorbed from a drug product and becomesavailable at the site of action. For example, bioavailability can bemeasured as the amount of API in the blood (serum or plasma) as afunction of time. Pharmacokinetic (PK) parameters such as AUC, C_(max),or T_(max) may be used to measure and assess bioavailability. For drugproducts that are not intended to be absorbed into the bloodstream,bioavailability may be assessed by measurements intended to reflect therate and extent to which the API or active ingredient or active moietybecomes available at the site of action.

The term “bioequivalent,” which has the meaning defined in 21 C.F.R. §320.1(e), refers to the absence of a significant difference in the rateand extent to which the API or active ingredient or active moiety inpharmaceutical equivalents or pharmaceutical alternatives becomesavailable at the site of drug action when administered at the same molardose under similar conditions in an appropriately designed study. Wherethere is an intentional difference in rate (e.g., in certain extendedrelease dosage forms), certain pharmaceutical equivalents oralternatives may be considered bioequivalent if there is no significantdifference in the extent to which the active ingredient or moiety fromeach product becomes available at the site of drug action. This appliesonly if the difference in the rate at which the active ingredient ormoiety becomes available at the site of drug action is intentional andis reflected in the proposed labeling, is not essential to theattainment of effective body drug concentrations on chronic use, and isconsidered medically insignificant for the drug. In practice, twoproducts are considered bioequivalent if the 90% confidence interval ofthe AUC, C_(max), or optionally T_(max) is within 80.00% to 125.00%.

The term “bio-identical,” “body-identical,” or “natural” used inconjunction with the hormones disclosed herein, means hormones thatmatch the chemical structure and effect of those that occur naturally orendogenously in the human body. An exemplary natural estrogen isestradiol.

The term “bio-identical hormone” or “body-identical hormone” refers toan active pharmaceutical ingredient that is structurally identical to ahormone naturally or endogenously found in the human body (e.g.,estradiol and progesterone).

The term “estradiol” refers to (17β)-estra-1,3,5(10)-triene-3,17-diol.Estradiol is also interchangeably called 17β-estradiol, oestradiol, orE2, and is found endogenously in the human body. As used herein,estradiol refers to the bio-identical or body-identical form ofestradiol found in the human body having the structure:

Estradiol is supplied in an anhydrous or hemi-hydrate form. For thepurposes of this disclosure, the anhydrous form or the hemihydrate formcan be substituted for the other by accounting for the water or lack ofwater according to well-known and understood techniques.

The term “solubilized estradiol” means that the estradiol or a portionthereof is solubilized or dissolved in the solubilizing agent(s) or theformulations disclosed herein. Solubilized estradiol may includeestradiol that is about 80% solubilized, about 85% solubilized, about90% solubilized, about 95% solubilized, about 96% solubilized, about 97%solubilized, about 98% solubilized, about 99% solubilized or about 100%solubilized. In some embodiments, the estradiol is “fully solubilized”with all or substantially all of the estradiol being solubilized ordissolved in the solubilizing agent. Fully solubilized estradiol mayinclude estradiol that is about 97% solubilized, about 98% solubilized,about 99% solubilized or about 100% solubilized. Solubility can beexpressed as a mass fraction (% w/w, which is also referred to as wt %).

The term “progesterone” refers to pregn-4-ene-3,20-dione. Progesteroneis also interchangeably called P4 and is found endogenously in the humanbody. As used herein, progesterone refers to the bio-identical orbody-identical form of progesterone found in the human body having thestructure:

The term “solubilized progesterone” means that the progesterone or aportion thereof is solubilized or dissolved in the solubilizing agent(s)or the formulations disclosed herein. In some embodiments, theprogesterone is “partially solubilized” with a portion of theprogesterone being solubilized or dissolved in the solubilizing agentand a portion of the progesterone being suspended in the solubilizingagent. Partially solubilized progesterone may include progesterone thatis about 1% solubilized, about 5% solubilized, about 10% solubilized,about 15% solubilized, about 20% solubilized, about 30% solubilized,about 40% solubilized, about 50% solubilized, about 60% solubilized,about 70% solubilized, about 80% solubilized, about 85% solubilized,about 90% solubilized or about 95% solubilized. In other embodiments,the progesterone is “fully solubilized” with all or substantially all ofthe progesterone being solubilized or dissolved in the solubilizingagent. Fully solubilized progesterone may include progesterone that isabout 97% solubilized, about 98% solubilized, about 99% solubilized orabout 100% solubilized. Solubility can be expressed as a mass fraction(% w/w, which is also referred to as wt %).

The terms “micronized progesterone” and “micronized estradiol,” as usedherein, include micronized progesterone and micronized estradiol havingan X50 particle size value below about 15 microns or having an X90particle size value below about 25 microns. The term “X50” means thatone-half of the particles in a sample are smaller in diameter than agiven number. For example, micronized progesterone having an X50 of 5microns means that, for a given sample of micronized progesterone,one-half of the particles have a diameter of less than 5 microns.Similarly, the term “X90” means that ninety percent (90%) of theparticles in a sample are smaller in diameter than a given number.

The term “glyceride” is an ester of glycerol (1,2,3-propanetriol) withacyl radicals of fatty acids and is also known as an acylglycerol. Ifonly one position of the glycerol molecule is esterified with a fattyacid, a “monoglyceride” or “monoacylglycerol” is produced; if twopositions are esterified, a “diglyceride” or “diacylglycerol” isproduced; and if all three positions of the glycerol are esterified withfatty acids, a “triglyceride” or “triacylglycerol” is produced. Aglyceride is “simple” if all esterified positions contain the same fattyacid; whereas a glyceride is “mixed” if the esterified positionscontained different fatty acids. The carbons of the glycerol backboneare designated sn-1, sn-2 and sn-3, with sn-2 being in the middle carbonand sn-1 and sn-3 being the end carbons of the glycerol backbone.

The term “solubilizing agent” refers to an agent or combination ofagents that solubilize an active pharmaceutical ingredient (e.g.,estradiol or progesterone). For example and without limitation, suitablesolubilizing agents include medium chain oils and other solvents andco-solvents that solubilize or dissolve an active pharmaceuticalingredient to a desirable extent. Solubilizing agents suitable for usein the formulations disclosed herein are pharmaceutical gradesolubilizing agents (e.g., pharmaceutical grade medium chain oils). Itwill be understood by those of skill in the art that other excipients orcomponents can be added to or mixed with the solubilizing agent toenhance the properties or performance of the solubilizing agent orresulting formulation. Examples of such excipients include, but are notlimited to, surfactants, emulsifiers, thickeners, colorants, flavoringagents, etc. In some embodiments, the solubilizing agent is a mediumchain oil and, in some other embodiments, the medium chain oil iscombined with a co-solvent(s) or other excipient(s).

The term “medium chain” is used to describe the aliphatic chain lengthof fatty acid containing molecules. “Medium chain” specifically refersto fatty acids, fatty acid esters, or fatty acid derivatives thatcontain fatty acid aliphatic tails or carbon chains that contain 6 (C6)to 14 (C14) carbon atoms, 8 (C8) to 12 (C12) carbon atoms, or 8 (C8) to10 (C10) carbon atoms.

The terms “medium chain fatty acid” and “medium chain fatty acidderivative” are used to describe fatty acids or fatty acid derivativeswith aliphatic tails (i.e., carbon chains) having 6 to 14 carbon atoms.Fatty acids consist of an unbranched or branched aliphatic tail attachedto a carboxylic acid functional group. Fatty acid derivatives include,for example, fatty acid esters and fatty acid containing molecules,including, without limitation, mono-, di- and triglycerides that includecomponents derived from fatty acids. Fatty acid derivatives also includefatty acid esters of ethylene or propylene glycol. The aliphatic tailscan be saturated or unsaturated (i.e., having one or more double bondsbetween carbon atoms). In some embodiments, the aliphatic tails aresaturated (i.e., no double bonds between carbon atoms). Medium chainfatty acids or medium chain fatty acid derivatives include those withaliphatic tails having 6-14 carbons, including those that are C6-C14,C6-C12, C8-C14, C8-C12, C6-C10, C8-C10, or others. Examples of mediumchain fatty acids include, without limitation, caproic acid, caprylicacid, capric acid, lauric acid, myristic acid, and derivatives thereof.

The term “oil,” as used herein, refers to any pharmaceuticallyacceptable oil, especially medium chain oils, and specifically excludingpeanut oil, that can suspend or solubilize bioidentical progesterone orestradiol, including starting materials or precursors thereof, includingmicronized progesterone or micronized estradiol as described herein.

The term “medium chain oil” refers to an oil wherein the composition ofthe fatty acid fraction of the oil is substantially medium chain (i.e.,C6 to C14) fatty acids, i.e., the composition profile of fatty acids inthe oil is substantially medium chain. As used herein, “substantially”means that between 20% and 100% (inclusive of the upper and lowerlimits) of the fatty acid fraction of the oil is made up of medium chainfatty acids, i.e., fatty acids with aliphatic tails (i.e., carbonchains) having 6 to 14 carbons. In some embodiments, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 85%, about 90% or about 95% ofthe fatty acid fraction of the oil is made up of medium chain fattyacids. Those of skill in the art that will readily appreciate that theterms “alkyl content” or “alkyl distribution” of an oil can be used inplace of the term “fatty acid fraction” of an oil in characterizing agiven oil or solubilizing agent, and these terms are usedinterchangeable herein. As such, medium chain oils suitable for use inthe formulations disclosed herein include medium chain oils wherein thefatty acid fraction of the oil is substantially medium chain fattyacids, or medium chain oils wherein the alkyl content or alkyldistribution of the oil is substantially medium chain alkyls (C6-C12alkyls). It will be understood by those of skill in the art that themedium chain oils suitable for use in the formulations disclosed hereinare pharmaceutical grade (e.g., pharmaceutical grade medium chain oils).Examples of medium chain oils include, for example and withoutlimitation, medium chain fatty acids, medium chain fatty acid esters ofglycerol (e.g., for example, mono-, di-, and triglycerides), mediumchain fatty acid esters of propylene glycol, medium chain fatty acidderivatives of polyethylene glycol, and combinations thereof.

The term “ECN” or “equivalent carbon number” means the sum of the numberof carbon atoms in the fatty acid chains of an oil, and can be used tocharacterize an oil as, for example, a medium chain oil or a long-chainoil. For example, tripalmitin (tripalmitic glycerol), which is a simpletriglyceride containing three fatty acid chains of 16 carbon atoms, hasan ECN of 3×16=48. Conversely, a triglyceride with an ECN=40 may have“mixed” fatty acid chain lengths of 8, 16 and 16; 10, 14 and 16; 8, 14and 18; etc. Naturally occurring oils are frequently “mixed” withrespect to specific fatty acids, but tend not to contain both long chainfatty acids and medium chain fatty acids in the same glycerol backbone.Thus, triglycerides with ECN's of 21-42 typically contain predominatelymedium chain fatty acids; while triglycerides with ECN's of greater than43 typically contain predominantly long chain fatty acids. For example,the ECN of corn oil triglyceride in the USP would be in the range of51-54. Medium chain diglycerides with ECN's of 12-28 will often containpredominately medium chain fatty chains, while diglycerides with ECN'sof 32 or greater will typically contain predominately long chain fattyacid tails. Monoglycerides will have an ECN that matches the chainlength of the sole fatty acid chain. Thus, monoglyceride ECN's in therange of 6-14 contain mainly medium chain fatty acids, andmonoglycerides with ECN's 16 or greater will contain mainly long chainfatty acids.

The average ECN of a medium chain triglyceride oil is typically 21-42.For example, as listed in the US Pharmacopeia (USP), medium chaintriglycerides have the following composition as the exemplary oil setforth in the table below:

Fatty-acid Tail Length % of oil Exemplary Oil 6 ≤2.0 2.0 8 50.0-80.070.0 10 20.0-50.0 25.0 12 ≤3.0 2.0 14 ≤1.0 1.0and would have an average ECN of3*[(6*0.02)+(8*0.70)+(10*0.25)+(12*0.02)+(14*0.01)]=25.8. The ECN of theexemplary medium chain triglycerides oil can also be expressed as arange (per the ranges set forth in the USP) of 24.9-27.0. For oils thathave mixed mono-, di-, and trigylcerides, or single and double fattyacid glycols, the ECN of the entire oil can be determined by calculatingthe ECN of each individual component (e.g., C8 monoglycerics, C8diglycerides, C10 monoglycerides, and C10 monoglycerides) and taking thesum of the relative percentage of the component multiplied by the ECNnormalized to a monoglyceride for each component. For example, the oilhaving C8 and C10 mono- and diglycerides shown in the table below has anECN of 8.3, and is thus a medium chain oil.

ECN as % of oil ECN as % of oil Fatty-acid % of (chain length) ×normalized to ChainLength oil (% in oil) monoglyceride C8 monoglyceride47     8 × 0.47 = 3.76  3.76 C10 monoglyceride 8    10 × 0.08 = 0.8 0.8C8 diglyceride 38 2 × (8 × 0.38) = 6.08 6.08/2 = 3.04 C10 diglyceride 72 × (10 × 0.07) = 1.4  1.4/2 = 0.7 OIL ECN 8.3 (normalized tomonoglycerides)Expressed differently, ECN can be calculated as each chain length in thecomposition multiplied by its relative percentage in the oil:(8*0.85)+(10*0.15)=8.3.

The term “excipients,” as used herein, refers to non-API ingredientssuch as solubilizing agents, anti-oxidants, oils, lubricants, and othersused in formulating pharmaceutical products.

The term “patient” or “subject” refers to an individual to whom thepharmaceutical composition is administered.

The term “pharmaceutical composition” refers to a pharmaceuticalcomposition comprising at least a solubilizing agent and estradiol. Asused herein, pharmaceutical compositions are delivered, for example viapessary (i.e., vaginal suppository), or absorbed vaginally.

The term “progestin” means any natural or man-made substance that haspharmacological properties similar to progesterone.

The term “reference listed drug product” (“RLD”) means VAGIFEM®(estradiol vaginal tablets) or ESTRACE® vaginal cream.

The terms “treat,” “treating,” and “treatment” refer to any indicia ofsuccess in the treatment or amelioration of an injury, disease, orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,disease, or condition more tolerable to the patient; slowing in the rateof degeneration or decline; or improving a patient's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subject parameters, including the results of a physicalexamination, neuropsychiatric examinations, or psychiatric evaluation.

The terms “atrophic vaginitis,” “vulvovaginal atrophy,” “vaginalatrophy,” and “VVA” are used herein interchangeably. The molecularmorphology of VVA is well known in the medical field.

INTRODUCTION

Provided herein are pharmaceutical compositions comprising solubilizedestradiol designed to be absorbed vaginally. The pharmaceuticalcompositions disclosed herein are designed to be absorbed and have theirtherapeutic effect locally, e.g., in vaginal or surrounding tissue.Further disclosed herein are data demonstrating efficacy of thepharmaceutical compositions disclosed, as well as methods relating tothe pharmaceutical compositions. Generally, the pharmaceuticalcompositions disclosed herein are useful in VVA, dysparuenia, and otherindications caused by decrease or lack of estrogen.

Additional aspects and embodiments of this disclosure include: providingincreased patient ease of use while potentially minimizing certain sideeffects from inappropriate insertion, minimizing incidence ofvulvovaginal mycotic infection compared to incidence of vulvovaginalmycotic infection due to usage of other vaginally applied estradiolproducts; and, improved side effect profile (e.g., pruritus) compared tothe reference drug: VAGIFEM® (estradiol vaginal tablets, Novo Nordisk;Princeton, N.J.).

Pharmaceutical Composition

Functionality

According to embodiments, the pharmaceutical compositions disclosedherein are alcohol-free or substantially alcohol-free. Thepharmaceutical compositions offer provide for improved patientcompliance because of improvements over the prior offering. According toembodiments, the pharmaceutical compositions disclosed herein areencapsulated in soft gelatin capsules, which improve comfort during use.According to embodiments, the pharmaceutical compositions aresubstantially liquid, which are more readily absorbed in the vaginaltissue, and also are dispersed over a larger surface area of the vaginaltissue.

Estradiol

According to embodiments, the pharmaceutical compositions disclosedherein are for vaginal insertion in a single or multiple unit dosageform. According to embodiments, the estradiol in the pharmaceuticalcompositions is at least about: 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% solubilized. According to embodiments and wherethe estradiol is not 100% solubilized, the remaining estradiol ispresent in a micronized (crystalline) form that is absorbable by thebody and retains biological functionality, either in its micronized formor in another form which the micronized form is converted to afteradministration.

According to embodiments, all or some of the estradiol is solubilized ina solubilizing agent during manufacturing process. According toembodiments, all or some of the estradiol is solubilized followingadministration (e.g., the micronized portion where the estradiol is not100% solubilized is solubilized in a body fluid after administration).According to embodiments, because the estradiol is solubilized, thesolubilizing agents taught herein, with or without additional excipientsother than the solubilizing agents, are liquid or semi-solid. To theextent the estradiol is not fully solubilized at the time ofadministration/insertion, the estradiol should be substantiallysolubilized at a body temperature (average of 37° C.) and, generally, atthe pH of the vagina (ranges from 3.8 to 4.5 in healthy patients; and4.6 to 6.5 in VVA patients).

According to embodiments, the estradiol can be added to thepharmaceutical compositions disclosed herein as estradiol, estradiolhemihydrate, or other grade estradiol forms used in pharmaceuticalcompositions or formulations.

According to embodiments, estradiol dosage strengths vary. Estradiol (orestradiol hemihydrate, for example, to the extent the water content ofthe estradiol hemihydrate is accounted for) dosage strength of is fromat least about 1 microgram (μg or μg) to at least about 50 μg. Specificdosage embodiments contain at least about: 1 μg, 2 μg, 3 μg, 4 μg, 5 μg,6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg,17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, 25 μg, 26 μg, 27μg, 28 μg, 29 μg, 30 μg, 31 μg, 32 μg, 33 μg, 34 μg, 35 μg, 36 μg, 37μg, 38 μg, 39 μg, 40 μg, 41 μg, 42 μg, 43 μg, 44 μg, 45 μg, 46 μg, 47μg, 48 μg, 49 μg, or 50 μg estradiol. According to embodiments, thepharmaceutical compositions contain at least about 2.5 μg; 4 μg 6.25 μg,7.5 μg, 12.5 μg, 18.75 μg of estradiol. According to embodiments, thepharmaceutical compositions contain from about 1 μg to about 10 μg, from3 μg to 7 μg, from about 7.5 μg to 12.5 μg, from about 10 μg to about 25μg, about 1 μg, about 2.5 μg, from about 23.5 μg to 27.5 μg, from about7.5 μg to 22.5 μg, from 10 μg to 25 μg of estradiol. The lowestclinically effective dose of estradiol is used for treatment of VVA andother indications set forth herein. In some embodiments, the estradioldosage is about 4 μg. In one embodiment, the estradiol dosage is about10 μg. In another embodiment, the estradiol dosage is about 25 μg.

Solvent System

According to embodiments, the solvent system that solubilizes theestradiol are medium chain fatty acid based solvents, together withother excipients. According to embodiments, the solvent system comprisesnon-toxic, pharmaceutically acceptable solvents, co-solvents,surfactants, and other excipients suitable for vaginal delivery orabsorption.

According to embodiments, oils having medium chain fatty acids as amajority component are used as solubilizing agents to solubilizeestradiol. According to embodiments, the solubilizing agents comprisemedium chain fatty acid esters (e.g., esters of glycerol, ethyleneglycol, or propylene glycol) or mixtures thereof. According toembodiments, the medium chain fatty acids comprise chain lengths from C6to C14. According to embodiments the medium chain fatty acids comprisechain lengths from C6 to C12. According to embodiments the medium chainfatty acids substantially comprise chain lengths from C8-C10. ECN's formedium chain oils will be in the range of 21-42 for triglycerides, 12-28for diglycerides, and 6-14 for monoglycerides.

According to embodiments, the medium chain fatty acids are saturated.According to embodiments, the medium chain fatty acids are predominantlysaturated, i.e., greater than about 60% or greater than about 75%saturated.

According to embodiments, estradiol is soluble in the solubilizing agentat room temperature, although it may be desirable to warm certainsolubilizing agents during manufacture to improve viscosity. Accordingto embodiments, the solubilizing agent is liquid at between roomtemperature and about 50° C., at or below 50° C., at or below 40° C., orat or below 30° C.

According to embodiments, the solubility of estradiol in the mediumchain oil, medium chain fatty acid, or solubilizing agent (oroil/surfactant) is at least about 0.01 wt %, 0.02 wt %, 0.05 wt %, 0.06wt %, 0.08 wt %, 0.1 wt %, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6wt %, 0.7 wt %, 0.8 wt %, 0.9 wt %, 1.0 wt %, or higher.

According to embodiments, medium chain solubilizing agents include, forexample and without limitation saturated medium chain fatty acids:caproic acid (C6), enanthic acid (C7), caprylic acid (C8), pelargonicacid (C9), capric acid (C10), undecylic acid (C11), lauric acid (C12),tridecylic acid (C13), or myristic acid (C14). According to embodiments,the solubilizing agent comprises oils made of these free medium chainfatty acids, oils of medium chain fatty acid esters of glycerin,propylene glycol, or ethylene glycol, or combinations thereof. Theseexamples comprise predominantly saturated medium chain fatty acids(i.e., greater than 50% of the fatty acids are medium chain saturatedfatty acids). According to embodiments, predominantly C6 to C12saturated fatty acids are contemplated. According to embodiments, thesolubilizing agent is selected from at least one of a solvent orco-solvent.

According to embodiments, glycerin based solubilizing agents include:mono-, di-, or triglycerides and combinations and derivatives thereof.Exemplary glycerin based solubilizing agents include MIGLYOLs®, whichare caprylic/capric triglycerides (SASOL Germany GMBH, Hamburg).MIGLYOLs includes MIGLYOL 810 (caprylic/capric triglyceride), MIGLYOL812 (caprylic/capric triglyceride), MIGLYOL 816 (caprylic/caprictriglyceride), and MIGLYOL 829 (caprylic/capric/succinic triglyceride).Other caprylic/capric triglyceride solubilizing agents are likewisecontemplated, including, for example: caproic/caprylic/capric/laurictriglycerides; caprylic/capric/linoleic triglycerides;caprylic/capric/succinic triglycerides. According to embodiments, CAPMULMCM, medium chain mono- and di-glycerides, is the solubilizing agent.Other and triglycerides of fractionated vegetable fatty acids, andcombinations or derivatives thereof can be the solubilizing agent,according to embodiments. For example, the solubilizing agent can be1,2,3-propanetriol (glycerol, glycerin, glycerine) esters of saturatedcoconut and palm kernel oil and derivatives thereof.

Ethylene and propylene glycols (which include polyethylene andpolypropylene glycols) solubilizing agents include: glyceryl mono- anddi-caprylates; propylene glycol monocaprylate (e.g., CAPMUL® PG-8 (theCAPMUL brands are owned by ABITEC, Columbus, Ohio)); propylene glycolmonocaprate (e.g., CAPMUL PG-10); propylene glycol mono- anddicaprylates; propylene glycol mono- and dicaprate; diethylene glycolmono ester (e.g., TRANSCUTOL®, 2-(2-Ethoxyethoxy)ethanol, GATIEFOSSÉSAS); and diethylene glycol monoethyl ether. Other combinations of mono-and di-esters of propylene glycol or ethylene glycol are expresslycontemplated are the solubilizing agent.

According to embodiments, the solubilizing agent comprises combinationsof mono- and di-propylene and ethylene glycols and mono-, di-, andtriglyceride combinations. According to embodiments, polyethylene glycolglyceride (GELUCIRE®, GATIEFOSSÉ SAS, Saint-Priest, France) can be usedherein as the solubilizing agent or as a surfactant. For example,GELUCIRE 44/14 (PEG-32 glyceryl laurate EP), a medium chain fatty acidesters of polyethylene glycol, is a polyethylene glycol glyceridecomposed of mono-, di- and triglycerides and mono- and diesters ofpolyethylene glycol.

According to embodiments, commercially available fatty acid glycerol andglycol ester solubilizing agents are often prepared from natural oilsand therefore may comprise components in addition to the fatty acidesters that predominantly comprise and characterize the solubilizingagent. Such other components may be, e.g., other fatty acid mono-, di-,and triglycerides; fatty acid mono- and diester ethylene or propyleneglycols, free glycerols or glycols, or free fatty acids, for example. Insome embodiments, when an oil/solubilizing agent is described herein asa saturated C₈ fatty acid mono- or diester of glycerol, the predominantcomponent of the oil, i.e., >50 wt % (e.g., >75 wt %, >85 wt % or >90 wt%) is caprylic monoglycerides and caprylic diglycerides. For example,the Technical Data Sheet by ABITEC for CAPMUL MCM C8 describes CAPMULMCM C8 as being composed of mono and diglycerides of medium chain fattyacids (mainly caprylic) and describes the alkyl content as ≤1% C6, ≥95%C8, ≤5% C10, and ≤1.5% C12 and higher.

For example, MIGLYOL 812 is a solubilizing agent that is generallydescribed as a C8-C10 triglyceride because the fatty acid composition isat least about 80% triglyceride esters of caprylic acid (C8) and capricacid (C10). However, it also comprises small amounts of other fattyacids, e.g., less than about 5% of caproic acid (C6), lauric acid (C12),and myristic acid (C14). The product information sheet for variousMIGLYOLs illustrate the various fatty acid components as follows:

Tests 810 812 818 829 840 Caproic acid max. 2.0 max. 2.0 max. 2 max. 2max. 2 (C6:0) Caprylic acid 65.0-80.0 50.0-65.0 45-65 45-55 65-80 (C8:0)Capric acid 20.0-35.0 30.0-45.0 30-45 30-40 20-35 (C10:0) Lauric acidmax. 2   max. 2   max. 3 max. 3 max. 2 (C12:0) Myristic acid max. 1.0max. 1.0 max. 1 max. 1 max. 1 (C14:0) Linoleic acid — — 2-5 — — (C18:2)Succinic acid — — — 15-20 — ECN 25.5-26.4 26.1-27   26.52-28.56 26-27.625.5-26.4

According to embodiments, anionic or non-ionic surfactants may be usedin pharmaceutical compositions containing solubilized estradiol. Ratiosof solubilizing agent(s) to surfactant(s) vary depending upon therespective solubilizing agent(s) and the respective surfactant(s) andthe desired physical characteristics of the resultant pharmaceuticalcomposition. For example and without limitation, CAPMUL MCM and anon-ionic surfactant may be used at ratios including 65:35, 70:30,75:25, 80:20, 85:15 and 90:10. Other non-limiting examples include:CAPMUL MCM and GELUCIRE 39/01 used in ratios including, for example andwithout limitation, 6:4, 7:3, and 8:2; CAPMUL MCM and GELUCIRE 43/01used in ratios including, for example and without limitation, 7:3, and8:2; CAPMUL MCM and GELUCIRE 50/13 used in ratios including, for exampleand without limitation, 7:3, and 8:2, and 9:1.

Other Excipients

According to embodiments, the pharmaceutical composition furthercomprises a surfactant. The surfactant can be a nonionic surfactant,cationic surfactant, anionic surfactant, or mixtures thereof. Suitablesurfactants include, for example, water-insoluble surfactants having ahydrophilic-lipophilic balance (HLB) value less than 12 andwater-soluble surfactants having a HLB value greater than 12.Surfactants that have a high HLB and hydrophilicity, aid the formationof oil-water droplets. The surfactants are amphiphilic in nature and arecapable of dissolving or solubilizing relatively high amounts ofhydrophobic drug compounds.

Non-limiting examples, include, Tween, Dimethylacetamide (DMA), Dimethylsulfoxide (DMSO), Ethanol, Glycerin, N-methyl-2-pyrrolidone (NMP), PEG300, PEG 400, Poloxamer 407, Propylene glycol, Phospholipids,Hydrogenated soy phosphatidylcholine (HSPC),Distearoylphosphatidylglycerol (DSPG),L-α-dimyristoylphosphatidylcholine (DMPC),L-α-dimyristoylphosphatidylglycerol (DMPG), Polyoxyl 35 castor oil(CREMOPHOR EL, CREMOPHOR ELP), Polyoxyl 40 hydrogenated castor oil(Cremophor RH 40), Polyoxyl 60 hydrogenated castor oil (CREMOPHOR RH60), Polysorbate 20 (TWEEN 20), Polysorbate 80 (TWEEN 80),d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), Solutol HS-15,Sorbitan monooleate (SPAN 20), PEG 300 caprylic/capric glycerides(SOFTIGEN 767), PEG 400 caprylic/capric glycerides (LABRASOL), PEG 300oleic glycerides (LABRAFIL M-1944CS), Polyoxyl 35 Castor oil (ETOCAS35), Glyceryl Caprylate (Mono- and Diglycerides) (IMWITOR), PEG 300linoleic glycerides (LABRAFIL M-2125CS), Polyoxyl 8 stearate (PEG 400monosterate), Polyoxyl 40 stearate (PEG 1750 monosterate), andcombinations thereof. Additionally, suitable surfactants include, forexample, polyoxyethylene derivative of sorbitan monolaurate such aspolysorbate, caprylcaproyl macrogol glycerides, polyglycolyzedglycerides, and the like.

According to embodiments, the non-ionic surfactant is selected from oneor more of glycerol and polyethylene glycol esters of long chain fattyacids, for example, lauroyl macrogol-32 glycerides or lauroylpolyoxyl-32 glycerides, commercially available as GELUCIRE, including,for example, GELUCIRE 39/01 (glycerol esters of saturated C12-C18 fattyacids), GELUCIRE 43/01 (hard fat NF/JPE) and GELUCIRE 50/13 (stearoylmacrogol-32 glycerides EP, stearoyl polyoxyl-32 glycerides NF, stearoylpolyoxylglycerides (USA FDA IIG)). These surfactants may be used atconcentrations greater than about 0.01%, and typically in variousamounts of about 0.01%-10.0%, 10.1%-20%, and 20.1%-30%. In someembodiments, surfactants may be used at concentrations of about 1% toabout 10% (e.g., about 1% to about 5%, about 2% to about 4%, about 3% toabout 8%).

According to embodiments, non-ionic surfactants include, for example andwithout limitation: one or more of oleic acid, linoleic acid, palmiticacid, and stearic acid. According to embodiments, non-ionic surfactantscomprise polyethylene sorbitol esters, including polysorbate 80, whichis commercially available under the trademark TWEEN® 80 (polysorbate 80)(Sigma Aldrich, St. Louis, Mo.). Polysorbate 80 comprises approximately60%-70% oleic acid with the remainder comprising primarily linoleicacids, palmitic acids, and stearic acids. Polysorbate 80 may be used inamounts ranging from about 5 to 50%, and according to embodiments, about30% of the pharmaceutical composition total mass.

According to embodiments, the non-ionic surfactant includes PEG-6palmitostearate and ethylene glycol palmitostearate, which are availablecommercially as TEFOSE® 63 (GATTEFOSSÉ SAS, Saint-Priest, France), whichcan be used with, for example, CAPMUL MCM having ratios of MCM to TEFOSE63 of, for example, 8:2 or 9:1. According to embodiments, othersolubilizing agents/non-ionic surfactants combinations include, forexample, MIGLYOL 812:GELUCIRE 50/13 or MIGLYOL 812:TEFOSE 63.

According to embodiments, the surfactant can be an anionic surfactant,for example: ammonium lauryl sulfate, dioctyl sodium sulfosuccinate,perfluoro-octane sulfonic acid, potassium lauryl sulfate, or sodiumstearate. Cationic surfactants are also contemplated.

According to embodiments, non-ionic or anionic surfactants can be usedalone with at least one solubilizing agent or can be used in combinationwith other surfactants. Accordingly, such surfactants, or any otherexcipient as set forth herein, may be used to solubilize estradiol. Thecombination of solubilizing agent, surfactant, and other excipientsshould be designed whereby the estradiol is absorbed into the vaginaltissue. According to embodiments, the pharmaceutical composition willresult in minimal vaginal discharge.

According to embodiments, the pharmaceutical composition furthercomprises at least one thickening agent. Generally, a thickening agentis added when the viscosity of the pharmaceutical composition resultsless than desirable absorption. According to embodiments, thesurfactant(s) disclosed herein may also provide thickening of thepharmaceutical composition that, upon release, will aid the estradiol inbeing absorbed by the vaginal mucosa while minimizing vaginal discharge.Examples of thickening agents include: hard fats; propylene glycol; amixture of hard fat EP/NF/JPE, glyceryl ricinoleate, ethoxylated fattyalcohols (ceteth-20, steareth-20) EP/NF (available as OVUCIRE® 3460,GATTEFOSSÉ, Saint-Priest, France); a mixture of hard fat EP/NF/JPE,glycerol monooleate (type 40) EP/NF (OVUCIRE WL 3264; a mixture of hardfat EP/NF/JPE, glyceryle monooleate (type 40) EP/NF (OVUCIRE WL 2944); anon-ionic surfactant comprising PEG-6 stearate, ethylene glycolpalmitostearate, and PEG-32 stearate; TEFOSE 63 or a similar product;and a mixture of various hard fats (WITEPSOL®, Sasol Germany GmbH,Hamburg, Germany). Other thickening agents such as the alginates,certain gums such as xanthan gums, agar-agar, iota carrageenans, kappacarrageenans, etc. Several other compounds can act as thickening agentslike gelatin, and polymers like HPMC, PVC, and CMC. According toembodiments, the viscosity of pharmaceutical compositions in accordancewith various embodiments may comprise from about 50 cps to about 1000cps at 25° C. A person of ordinary skill in the art will readilyunderstand and select from suitable thickening agents.

According to embodiments, the thickening agent is a non-ionicsurfactant. For example, polyethylene glycol saturated or unsaturatedfatty acid ester or diester is the non-ionic surfactant thickeningagent. In embodiments, the non-ionic surfactant comprises a polyethyleneglycol long chain (C16-C20) fatty acid ester and further comprises anethylene glycol long chain fatty acid ester, such as PEG-fatty acidesters or diesters of saturated or unsaturated C16-C18 fatty acids,e.g., oleic, lauric, palmitic, and stearic acids. In embodiments, thenon-ionic surfactant comprises a polyethylene glycol long chainsaturated fatty acid ester and further comprises an ethylene glycol longchain saturated fatty acid ester, such as PEG- and ethylene glycol-fattyacid esters of saturated C16-C18 fatty acids, e.g., palmitic and stearicacids. Such non-ionic surfactant can comprise PEG-6 stearate, ethyleneglycol palmitostearate, and PEG-32 stearate, such as but not limited toTEFOSE 63.

According to embodiments, the non-ionic surfactant used as a thickeningagent is not hydrophilic and has good emulsion properties. Anillustrative example of such surfactant is TEFOSE 63, which has ahydrophilic-lipophilic balance (HLB) value of about 9-10.

According to embodiments, the pharmaceutical composition furthercomprises one or more mucoadherent agents to improve vaginal absorptionof the estradiol. For example, a mucoadherent agent can be present toaid the pharmaceutical composition with adherence to the mucosa uponactivation with water. According to embodiments, polycarbophil is themucoadherent agent. According to embodiments, other mucoadherent agentsinclude, for example and without limitation: poly (ethylene oxide)polymers having a molecular weight of from about 100,000 to about900,000; chitosans carbopols including polymers of acrylic acidcrosslinked with allyl sucrose or allyl pentaerythritol; polymers ofacrylic acid and C10-C30 alkyl acrylate crosslinked with allylpentaerythritol; carbomer homopolymer or copolymer that contains a blockcopolymer of polyethylene glycol and a long chain alkyl acid ester; andthe like. According to embodiments, various hydrophilic polymers andhydrogels may be used as the mucoadherent agent. According to certainembodiments, the polymers or hydrogels can swell in response to contactwith vaginal tissue or secretions, enhancing moisturizing andmucoadherent effects. The selection and amount of hydrophilic polymermay be based on the selection and amount of solubilizing agent. In someembodiments, the pharmaceutical composition includes a hydrophilicpolymer but optionally excludes a gelling agent. In embodiments having ahydrogel, from about 5% to about 10% of the total mass may comprise thehydrophilic polymer. In further embodiments, hydrogels may be employed.A hydrogel may comprise chitosan, which swell in response to contactwith water. In various embodiments, a cream pharmaceutical compositionmay comprise PEG-90M. In some embodiments, a mucoadherent agent ispresent in the pharmaceutical formulation, in the soft gel capsule, orboth.

According to embodiments, the pharmaceutical compositions include one ormore thermoreversible gels, typically of the hydrophilic natureincluding for example and without limitation, hydrophilic sucrose andother saccharide-based monomers (U.S. Pat. No. 6,018,033, which isincorporated by reference).

According to embodiments, the pharmaceutical composition furthercomprises a lubricant. In some embodiments, a lubricant can be presentto aid in formulation of a dosage form. For example, a lubricant may beadded to ensure that capsules or tablets do not stick to one anotherduring processing or upon storage. Any suitable lubricant may be used.For example, lecithin, which is a mixture of phospholipids, is thelubricant.

According to embodiments, the pharmaceutical composition furthercomprises an antioxidant. Any suitable anti-oxidant may be used. Forexample, butylated hydroxytoluene, butylated hydroxyanisole, and VitaminE TPGS.

According to embodiments, the pharmaceutical composition comprises about20% to about 80% solubilizing agent by weight, about 0.1% to about 5%lubricant by weight, and about 0.01% to about 0.1% antioxidant byweight.

The choice of excipient will depend on factors such as, for example, theeffect of the excipient on solubility and stability. Additionalexcipients used in various embodiments may include colorants andpreservatives. Examples of colorants include FD&C colors (e.g., blue No.1 and Red No. 40), D&C colors (e.g., Yellow No. 10), and opacifiers(e.g., Titanium dioxide). According to embodiments, colorants, compriseabout 0.1% to about 2% of the pharmaceutical composition by weight.According to embodiments, preservatives in the pharmaceuticalcomposition comprise methyl and propyl paraben, in a ratio of about10:1, and at a proportion of about 0.005% and 0.05% by weight.

Generally, the solubilizing agents, excipients, other additives used inthe pharmaceutical compositions described herein, are non-toxic,pharmaceutically acceptable, compatible with each other, and maintainstability of the pharmaceutical composition and the various componentswith respect to each other. Additionally, the combination of variouscomponents that comprise the pharmaceutical compositions will maintainwill result in the desired therapeutic effect when administered to asubject.

Solubility of Estradiol

According to embodiments, solubilizing agents comprising mixtures ofmedium chain fatty acid glycerides, e.g., C6-C12, C8-C12, or C8-C10fatty acid mono- and diglycerides or mono-, di-, and triglyceridesdissolve estradiol. As illustrated in the Examples, good results wereobtained with solubilizing agents that are predominantly a mixture ofC8-C10 saturated fatty acid mono- and diglycerides, or medium chaintriglycerides (e.g., Miglyol 810 or 812). Longer chain glycerides appearto be not as well suited for dissolution of estradiol.

A solubilizing agent comprising propylene glycol monocaprylate (e.g.,CAPRYOL) and 2-(2-Ethoxyethoxy)ethanol (e.g., TRANSCUTOL) solubilizedestradiol well.

Manufacture of the Pharmaceutical Composition

According to embodiments, the pharmaceutical composition is prepared viablending estradiol with a pharmaceutically acceptable solubilizingagent, including for example and without limitation, at least one mediumchain fatty acid such as medium chain fatty acids consisting of at leastone mono-, di-, or triglyceride, or derivatives thereof, or combinationsthereof. According to embodiments, the pharmaceutical composition alsocomprises at least one glycol or derivatives thereof or combinationsthereof or combinations of at least one glyceride and glycol. Theglycol(s) may be used as solubilizing agents or to adjust viscosity and,thus, may be considered thickening agents, as discussed further herein.Optionally added are other excipients including, for example and withoutlimitation, anti-oxidants, lubricants, and the like. According toembodiments, the pharmaceutical composition comprises sufficientsolubilizing agent to fully solubilize the estradiol. It is expresslyunderstood, however, the other volumes of solubilizing agent can be useddepending on the level of estradiol solubilization desired. Persons ofordinary skill in the art will know and understand how to determine thevolume of solubilizing agent and other excipients depending on thedesired percent of estradiol to be solubilized in the pharmaceuticalcomposition.

In illustrative embodiments, GELUCIRE 44/14 (lauroyl macrogol-32glycerides EP, lauroyl polyoxyl-32 glycerides NF, lauroylpolyoxylglycerides (USA FDA IIG)) is heated to about 65° C. and CAPMULMCM is heated to about 40° C. to facilitate mixing of the oil andnon-ionic surfactant, although such heating is not necessary to dissolvethe estradiol.

Specific Examples disclosed herein provide additional principles andembodiments illustrating the manufactures of the pharmaceuticalcompositions disclosed herein.

Delivery Vehicle

Generally, the pharmaceutical compositions described herein deliveredintravaginally inside of a delivery vehicle, for example a capsule.According to embodiments, the capsules are soft capsules made ofmaterials well known in the pharmaceutical arts, for example, gelatin.However, according to embodiments, the delivery vehicle is integral withthe pharmaceutical composition (i.e., the pharmaceutical composition isthe delivery vehicle). In such embodiments the pharmaceuticalcompositions is a gel, cream, ointment, tablet, or other preparationthat is directly applied and absorbed vaginally.

According to embodiments, pharmaceutical compositions disclosed hereinare contained in capsules, such as soft gelatin capsules. According toembodiments, the capsules contain one or more of the following:hydrophilic gel-forming bioadhesive (e.g., mucoadhesive) agents; alipophilic agent; a gelling agent for the lipophilic agent, or ahydrodispersible agent. According to embodiments, the hydrophilicgel-forming bioadhesive agent is carboxyvinylic acid;hydroxypropylcellulose; carboxymethylcellulose; gelatin; xanthane gum;guar gum; aluminum silicate; or mixtures thereof. According toembodiments, the lipophilic agent is a liquid triglyceride; solidtriglyceride (e.g., with a melting point of about 35° C.); carnauba wax;cocoa butter; or mixtures thereof. According to embodiments, the gellingagent is a hydrophobic colloidal silica. According to embodiments, thehydrodispersible agent is: polyoxyethylene glycol; polyoxyethyleneglycol 7-glyceryl-cocoate; or mixtures thereof.

According to embodiments, the delivery vehicle is designed for ease ofinsertion. According to embodiments, the delivery vehicle is sizedwhereby it can be comfortably inserted into the vagina. According toembodiments, the delivery vehicle is prepared in a variety ofgeometries. For example, the delivery vehicle is shaped as a tear drop,a cone with frustoconical end, a cylinder, a cylinder with larger “cap”portion, or other shapes suitable for and that ease insertion into thevagina. According to embodiments, delivery vehicle is used in connectionwith an applicator. According to other embodiments, delivery vehicle isinserted digitally.

With reference to FIG. 2, delivery vehicle 200 comprises pharmaceuticalcomposition 202 and capsule 204. Width 208 represents the thickness ofcapsule 204, for example about 0.108 inches. The distance from one endof delivery vehicle 200 to another is represented by distance 206, forexample about 0.690 inches. The size of delivery vehicle 200 may also bedescribed by the arc swept by a radius of a given length. For example,arc 210, which is defined by the exterior of gelatin 204, is an arcswept by a radius of about 0.189 inches. Arc 212, which is defined bythe interior of capsule 204, is an arc swept by a radius of about 0.0938inches. Arc 214, which is defined by the exterior of gelatin 204opposite arc 210, is an arc swept by a radius of about 0.108 inches.Suitable capsules of other dimensions may be provided. According toembodiments, capsule 204 has dimensions the same as or similar to theratios as provided above relative to each other.

According to embodiments, the delivery vehicle is designed to remainingin the vagina until the pharmaceutical compositions are released.According to embodiments, delivery vehicle dissolves intravaginally andis absorbed into the vaginal tissue with the pharmaceutical composition,which minimizes vaginal discharge. In such embodiments, deliverymechanism is made from constituents that are non-toxic, for example,gelatin.

Design Factors for Vaginally Inserted Pharmaceutical Compositions

According to embodiments, the pharmaceutical composition is designed tomaximize favorable characteristics that lead to patient compliance(patients that discontinue treatment prior to completion of theprescribed course of therapy), without sacrificing efficacy. Favorablecharacteristics include, for example, lack of or reduction of irritationrelative to other hormone replacement pessaries, lack of or reduction invaginal discharge of the pharmaceutical composition and delivery vehiclerelative to other hormone replacement pessaries, lack of or reduction ofpharmaceutical composition or delivery vehicle residue inside thevagina, ease of administration compared to other hormone replacementpessaries, or improved efficacy of drug product relative to otherwisesimilar pharmaceutical compositions.

According to embodiments, the pharmaceutical composition isnon-irritating or minimizes irritation. Patient irritation comprisespain, pruritis (itching), soreness, excessive discharge, swelling, orother similar conditions. Patient irritation results in poor compliance.Non-irritating or reduced irritation pharmaceutical compositions aremeasured relative to competing hormone pessaries, including tablets,creams, or other intravaginal estrogen delivery forms.

According to embodiments, the pharmaceutical compositions does notresult in systemic exposure (e.g., blood circulation of estradiol),which improves safety. According to other embodiments, thepharmaceutical compositions disclosed herein result in significantlyreduced systemic exposure (e.g., blood circulation of estradiol) whencompared to RLDs.

According to embodiments, the pharmaceutical composition does not leaveresidue inside the vagina. Rather, the pharmaceutical composition anddelivery vehicle are substantially absorbed or dispersed withoutresulting in unabsorbed residue or unpleasant sensations of non-absorbedor non-dispersed drug product. Measurement of lack of residue isrelative to other vaginally inserted products or can be measuredobjectively with inspection of the vaginal tissues. For example, certainother vaginally inserted products contain starch which can result ingreater discharge from the vagina following administration than. In someembodiments, the pharmaceutical compositions provided herein provide alower amount, duration, or frequency of discharge followingadministration compared to other vaginally inserted products (e.g.,compressed tablets).

According to embodiments, the pharmaceutical composition improvesvaginal discharge compared to other pessaries, including pessaries thatdeliver hormones. Ideally, vaginal discharge is eliminated, minimized,or improved compared to competing products.

According to embodiments, the pharmaceutical compositions disclosedherein are inserted digitally. According to embodiments, thepharmaceutical compositions are digitally inserted approximately twoinches into the vagina without a need for an applicator. According toembodiments, the pharmaceutical compositions are designed to be alsoinserted with an applicator, if desired. According to some embodiments,because the site of VVA is in the proximal region of the vagina (towardsthe vaginal opening), the pharmaceutical compositions disclosed hereinare designed to be inserted in the proximal portion of the vagina.

Through extensive experimentation, various medium chain fatty acidesters of glycerol and propylene glycol demonstrated one or morefavorable characteristics for development as a human drug product.According to embodiments, the solubilizing agent was selected from atleast one of a solvent or co-solvent. Suitable solvents and co-solventsinclude any mono-, di- or triglyceride and glycols, and combinationsthereof.

According to embodiments, the pharmaceutical composition is deliveredvia a gelatin capsule delivery vehicle. According to these embodiments,the pharmaceutical composition is a liquid pharmaceutical composition.According to embodiments, the delivery vehicle is a soft capsule, forexample a soft gelatin capsule. Thus, the pharmaceutical composition ofsuch embodiments is encapsulated in the soft gelatin capsule or othersoft capsule.

According to embodiments, the pharmaceutical composition comprisesestradiol that is at least about 80% solubilized in a solubilizing agentcomprising one or more C6 to C14 medium chain fatty acid mono-, di-, ortriglycerides and, optionally, a thickening agent. According toembodiments, the pharmaceutical composition comprises estradiol that isat least about 80% solubilized one or more C6 to C12 medium chain fattyacid mono-, di-, or triglycerides, e.g., one or more C6 to C14triglycerides, e.g., one or more C6 to C12 triglycerides, such as one ormore C8-C10 triglycerides. These embodiments specifically contemplatethe estradiol being at least 80% solubilized. These embodimentsspecifically contemplate the estradiol being at least 90% solubilized.These embodiments specifically contemplate the estradiol being at least95% solubilized. These embodiments specifically contemplate theestradiol being fully solubilized.

As noted above, liquid pharmaceutical compositions are liquid at roomtemperature or at body temperature. For example, in some embodiments, apharmaceutical composition provided herein is a liquid formulationcontained within a soft gel capsule. Gels, hard fats, or other solidforms that are not liquid at room or body temperature are less desirablein embodiments of the pharmaceutical composition that are liquid.

The thickening agent serves to increase viscosity, e.g., up to about10,000 cP (10,000 mPa-s), typically to no more than about 5000 cP, andmore typically to between about 50 and 1000 cP. In embodiments, thenon-ionic surfactant, e.g., GELUCIRE or TEFOSE, may be solid at roomtemperature and require melting to effectively mix with the solubilizingagent. However, in these embodiments, the resultant pharmaceuticalcomposition remains liquid, albeit with greater viscosity, not solid.

According to embodiments, the pharmaceutical composition comprisesestradiol, the medium chain solubilizing agent, and the thickening agentas the ingredients delivered via a soft capsule delivery vehicle. Otheringredients, e.g., colorants, antioxidants, preservatives, or otheringredients may be included as well. However, the addition of otheringredients should be in amounts that do not materially change thesolubility of the estradiol, the pharmacokinetics of the pharmaceuticalcomposition, or efficacy of the pharmaceutical composition. Otherfactors that should be considered when adjusting the ingredients of thepharmaceutical composition include the irritation, vaginal discharge,intravaginal residue, and other relevant factors, for example those thatwould lead to reduced patient compliance. Other contemplated ingredientsinclude: oils or fatty acid esters, lecithin, mucoadherent agents,gelling agents, dispersing agents, or the like.

Methods

According to embodiments, the pharmaceutical compositions disclosedherein can be used for the treatment of WA, including the treatment ofat least one WA symptom including: vaginal dryness, vaginal or vulvarirritation or itching, dysuria, dysparuenia, and vaginal bleedingassociated with sexual activity, among others. According to embodimentsthe methods of treatment are generally applicable to females.

According to embodiments, the pharmaceutical compositions disclosedherein can be used for the treatment of estrogen-deficient urinarystates. According to embodiments, the pharmaceutical compositionsdisclosed herein can be used for the treatment of dysparuenia, orvaginal bleeding associated with sexual activity.

According to embodiments, treatment of the VVA, estrogen-deficienturinary states, and dysparuenia and vaginal bleeding associated withsexual activity occurs by administering the pharmaceutical compositionsintravaginally. According to embodiments where the delivery vehicle is acapsule, the patient obtains the capsule and inserts the capsule intovagina, where the capsule dissolves and the pharmaceutical compositionis releases into the vagina where it is absorbed into the vaginaltissue. In some embodiments, the pharmaceutical composition iscompletely absorbed into the vaginal tissue. In some embodiments, thepharmaceutical composition is substantially absorbed into the vaginaltissue (e.g., at least about 80% by weight, at least about 85% byweight, at least about 90% by weight, at least about 95% by weight, atleast about 97% by weight, at least about 98% by weight, or at leastabout 99% by weight of the composition is absorbed). According toembodiments, the capsule is inserted about two inches into the vaginadigitally, however the depth of insertion is generally any depth thatallows for adsorption of substantially all of the pharmaceuticalcomposition. According to embodiments, the capsule can also be appliedusing an applicator that deposits the capsule at an appropriate vaginaldepth as disclosed herein.

According to embodiments where the pharmaceutical composition is acream, gel, ointment, or other similar preparation, the pharmaceuticalcomposition is applied digitally, as is well known and understood in theart.

Upon release of the pharmaceutical composition in the vagina, estradiolis locally absorbed. For example, following administration of thepessary to the proximal region of the vagina of a patient provides atherapeutically effective concentration of estradiol over 24 hours inthe proximal region of the vagina.

According to embodiments, the timing of administration of thepharmaceutical composition of this disclosure may be conducted by anysafe means as prescribed by an attending physician. According toembodiments, a patient will administer the pharmaceutical composition(e.g., a capsule) intravaginally each day for 14 days, then twice weeklythereafter.

According to embodiments, the pharmaceutical compositions are vaginallyadministered with co-administration of an orally administeredestrogen-based (or progestin-based or progestin- and estrogen-based)pharmaceutical drug product, or patch, cream, gel, spray, transdermaldelivery system or other parenterally-administered estrogen-basedpharmaceutical drug product, each of which can include natural,bio-similar, or synthetic or other derived estrogens or progestins.According to embodiments, modulation of circulating estrogen levelsprovided via the administration of the pharmaceutical compositionsdisclosed herein, if any, are not intended to be additive to anyco-administered estrogen product and its associated circulating bloodlevels. According to other embodiments, co-administrated estrogenproducts are intended to have an additive effect as would be determinedby the patient physician.

According to embodiments, the efficacy and safety of the pharmaceuticalcompositions described herein in the treatment of the symptoms of VVAmay be determined. According to embodiments, the size, effect, cytology,histology, and variability of the VVA may be determined using variousendpoints to determine efficacy and safety of the pharmaceuticalcompositions described herein or as otherwise accepted in the art, atpresent or as further developed. On source of endpoints is with the USFood and Drug Administration's (FDA) published guidelines for treatmentof VVA with estradiol.

Measurement of Efficacy

According to embodiments, administration of the pharmaceuticalcompositions described herein resulted in treatment of the WA, as wellas improvement of one or more of the associated symptoms. Patients withWA experience shrinking of the vaginal canal in both length and diameterand the vaginal canal has fewer glycogen-rich vaginal cells to maintainmoisture and suppleness. In addition, the vaginal wall can become thin,pale, dry, or sometimes inflamed (atrophic vaginitis). These changes canmanifest as a variety of symptoms collectively referred to as VVA. Suchsymptoms include, without limitations, an increase in vaginal pH;reduction of vaginal epithelial integrity, vaginal secretions, orepithelial surface thickness; pruritis; vaginal dryness; dyspareunia(pain or bleeding during sexual intercourse); urinary tract infections;or a change in vaginal color. According to embodiments, efficacy ismeasured as a reduction of vulvar and vaginal atrophy in a patient backto premenopausal conditions. According to embodiments, the change ismeasured as a reduction in the severity of one or more atrophic effectsmeasured at baseline (screening, Day 1) and compared to a measurementtaken at Day 15 (end of treatment). Severity of the atrophic effect maybe measured using a scale of 0 to 3 where, for example, none=0, mild=1,moderate=2, or severe=3. Such scoring is implemented to evaluate thepre-treatment condition of patients; to determine the appropriate courseof a treatment regime; such as dosage, dosing frequency, and duration,among others; and post-treatment outcomes.

One of the symptoms of VVA is increased vaginal pH. In further aspectsof this disclosure, treatment with the pharmaceutical compositionsdescribed herein resulted in a decrease in vaginal pH. A decrease invaginal pH is measured as a decrease from the vaginal pH at baseline(screening) to the vaginal pH at Day 15, according to embodiments. Insome embodiments, a pH of 5 or greater may be associated with WA. Insome embodiments, pH is measured using a pH indicator strip placedagainst the vaginal wall. In some embodiments, a change in vaginal pH isa change in a patient's vaginal pH to a pH of less than about pH 5.0. Insome embodiments, a subject's vaginal pH may be less than about pH 4.9,pH 4.8, pH 4.7, pH 4.6, pH 4.5, pH 4.4, pH 4.3, pH 4.2, pH 4.1, pH 4.0,pH 3.9, pH 3.8, pH 3.7, pH 3.6, or pH 3.5.

According to embodiments, treatment with the pharmaceutical compositionsdescribed herein resulted in improvements in the vaginal MaturationIndex. The Maturation Index is measured as a change in cell composition.According to embodiments and as related to VVA, a change in cellcomposition is measured as the change in percent of composition oramount of parabasal vaginal cells, intermediate cells, and superficialvaginal cells, such as a change in the composition or amount ofparabasal vaginal cells compared with or, relative to, a change insuperficial vaginal cells. A subject having VVA symptoms often has anincreased number of parabasal cells and a reduced number of superficialcells (e.g., less than about 5%) compared with women who do not sufferfrom VVA. Conversely, a subject having decreasing VVA symptoms, or asotherwise responding to treatment, may demonstrate an improvement in theMaturation Index, specifically a decrease in the amount of parabasalcells or an increase in the amount of superficial cells compared tobaseline (screening). In embodiments, a decrease in parabasal cells ismeasured as a reduction in the percent of parabasal cells; the percentreduction may be at least about an 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% reduction in the number ofparabasal cells. In embodiments, a percent reduction may be at leastabout a 54% reduction in the number of parabasal cells. In embodiments,an increase in superficial cells is measured as an increase in thepercent of superficial cells; the percent increase in superficial cellsmay be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%increase in the number of superficial cells. In further embodiments, apercent increase may be at least about a 35% increase in the number ofsuperficial cells.

In some embodiments, an improvement in the Maturation Index is assessedas a change over time. For example, as a change in cell compositionmeasured at a baseline (screening) at Day 1 compared to the cellcomposition measured at Day 15. The change in cell composition may alsobe assessed as a change in the amount of parabasal cells over time,optionally in addition to measuring changes in parabasal cells andsuperficial cells as described above. Such cells may be obtained fromthe vaginal mucosal epithelium through routine gynecological examinationand examined by means of a vaginal smear.

In various further aspects of this disclosure, treatment with thepharmaceutical compositions described herein resulted in any of: anincrease in superficial cells; a decrease in parabasal cells; and anincrease in intermediate cells.

In further aspects of this disclosure, samples may be collected todetermine hormone levels, in particular, estradiol levels. In someembodiments, blood samples may be taken from a subject and the level ofestradiol measured (pg/ml). In some embodiments, estradiol levels may bemeasured at 0 hours (for example, at time of first treatment), at 1 hour(for example, post first treatment), at 3 hours, and at 6 hours. In someembodiments, samples may be taken at day 8 (for example, post firsttreatment) and at day 15 (for example, one day post the last treatmenton day 14). In some embodiments, descriptive statistics of plasmaestradiol concentrations at each sampling time and observed C_(max) andT_(max) values may be measured and the AUC calculated.

In some embodiments, a pessary can comprise about 25 μg of estradiol. Insuch cases, administration of the pessary to a patient can provide, in aplasma sample from the patient, parameters including one or moreparameters selected from: 1) a corrected geometric mean peak plasmaconcentration (C_(max)) of estradiol of about 19 pg*hr/ml to about 29pg*hr/ml (e.g., 19.55 pg*hr/ml to about 28.75 pg*hr/ml); or 2) acorrected geometric mean area under the curve (AUC)₀₋₂₄ of estradiol ofabout 75 pg*hr/ml to about 112 pg*hr/ml (e.g., 75.82 pg*hr/ml to about111.50). In some embodiments, administration of the pessary to a patientprovides, in a plasma sample from the patient, one or more parametersselected from: 1) a corrected geometric mean peak plasma concentration(C_(max)) of estrone of about 9 pg*hr/ml to about 14 pg*hr/ml (e.g.,9.17 pg*hr/ml to about 13.49 pg*hr/ml); and 2) a corrected geometricmean area under the curve (AUC)₀₋₂₄ of estrone of about 43 pg*hr/ml toabout 65 pg*hr/ml (e.g., 43.56 pg*hr/ml to about 64.06 pg*hr/ml). Insome embodiments, administration of the pessary to a patient provides,in a plasma sample from the patient, provides one or more parametersselected from: 1) a corrected geometric mean peak plasma concentration(C_(max)) of estrone sulfate of about 416 pg*hr/ml to about 613 pg*hr/ml(e.g., 416.53 pg*hr/ml to about 612.55 pg*hr/ml); and 2) a correctedgeometric mean area under the curve (AUC)₀₋₂₄ of estrone sulfate ofabout 3598 pg*hr/ml to about 5291 pg*hr/ml (e.g., 3598.04 pg*hr/ml toabout 5291.24 pg*hr/ml).

In some embodiments, a pessary can comprise about 10 μg of estradiol. Insuch cases, administration of the pessary to a patient can provide, in aplasma sample from the patient, one or more parameters selected from: 1)a corrected geometric mean peak plasma concentration (C_(max)) ofestradiol of about 12 pg*hr/ml to about 18 pg*hr/ml (e.g., 12.22pg*hr/ml to about 17.98 pg*hr/ml); 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estradiol of about 42 pg*hr/ml to about 63pg*hr/ml (e.g., 42.18 pg*hr/ml to about 62.02 pg*hr/ml); and 3) acorrected geometric mean time to peak plasma concentration (T_(max)) ofestradiol of about 1 hrs to about 3 hrs (e.g., 1.49 hrs to about 2.19hrs). In some embodiments, administration of the pessary to a patientprovides, in a plasma sample from the patient, one or more parametersselected from: 1) a corrected geometric mean peak plasma concentration(C_(max)) of estrone of about 4 pg*hr/ml to about 7 pg*hr/ml (e.g., 4.38pg*hr/ml to about 6.44 pg*hr/ml); 2) a corrected geometric mean areaunder the curve (AUC)₀₋₂₄ of estrone of about 20 pg*hr/ml to about 31pg*hr/ml (e.g., 20.60 pg*hr/ml to about 30.30 pg*hr/ml); and 3) acorrected geometric mean time to peak plasma concentration (T_(max)) ofestrone of about 4 hrs to about 8 hrs (e.g., 4.99 hrs to about 7.34hrs). In some embodiments, administration of the pessary to a patientprovides, in a plasma sample from the patient, one or more parametersselected from: 1) a corrected geometric mean peak plasma concentration(C_(max)) of estrone sulfate of about 10 pg*hr/ml to about 16 pg*hr/ml(e.g., 10.34 pg*hr/ml to about 15.20 pg*hr/ml); 2) a corrected geometricmean area under the curve (AUC)₀₋₂₄ of estrone sulfate of about 56pg*hr/ml to about 84 pg*hr/ml (e.g., 56.61 pg*hr/ml to about 83.25pg*hr/ml); and 3) a corrected geometric mean time to peak plasmaconcentration (T_(max)) of estrone sulfate of about 4 hrs to about 7 hrs(e.g., 4.67 hrs to about 6.86 hrs).

In some embodiments, a pessary can comprise about 4 μg of estradiol. Insuch cases, administration of the pessary to a patient can provide, in aplasma sample from the patient, one or more parameters selected from: 1)a corrected geometric mean peak plasma concentration (C_(max)) ofestradiol of about 4 pg*hr/ml to about 8 pg*hr/ml; 2) a correctedgeometric mean area under the curve (AUC)₀₋₂₄ of estradiol of about 16pg*hr/ml to about 26 pg*hr/ml; and 3) a corrected geometric mean time topeak plasma concentration (T_(max)) of estradiol of about 0.25 hrs toabout 2 hrs. In some embodiments, administration of the pessary to apatient provides, in a plasma sample from the patient, one or moreparameters selected from: 1) a corrected geometric mean peak plasmaconcentration (C_(max)) of estrone of about 1 pg*hr/ml to about 3pg*hr/ml; 2) a corrected geometric mean area under the curve (AUC)₀₋₂₄of estrone of about 8 pg*hr/ml to about 13 pg*hr/ml; and 3) a correctedgeometric mean time to peak plasma concentration (T_(max)) of estrone ofabout 1 hrs to about 4 hrs. In some embodiments, administration of thepessary to a patient provides, in a plasma sample from the patient, oneor more parameters selected from: 1) a corrected geometric mean peakplasma concentration (C_(max)) of estrone sulfate of about 4 pg*hr/ml toabout 7 pg*hr/ml; 2) a corrected geometric mean area under the curve(AUC)₀₋₂₄ of estrone sulfate of about 22 pg*hr/ml to about 34 pg*hr/ml;and 3) a corrected geometric mean time to peak plasma concentration(T_(max)) of estrone sulfate of about 1 hrs to about 3 hrs.

A pharmaceutical composition provided herein can result in substantiallylocal delivery of estradiol. For example, plasma concentrations ofestradiol, estrone, and estrone sulfate measured in the plasma of apatient following administration of a pharmaceutical composition asprovided herein be statistically similar to those measured followingadministration of a placebo formulation (i.e. a similar formulationlacking the estradiol). Accordingly, in some embodiments, the plasmaconcentrations of estradiol, estrone, or estrone sulfate measuredfollowing administration of a pharmaceutical composition provided hereinmay be low compared to RLD formulations.

In some embodiments, a pessary can include about 1 μg to about 25 μg ofestradiol. Upon administration the pessary to a patient, a plasma samplefrom the patient can provide a corrected geometric mean peak plasmaconcentration (C_(max)) of estradiol that is less than about 30pg*hr/ml. For example, administration of the pessary to a patientprovides a corrected geometric mean peak plasma concentration (C_(max))of estradiol that is less than about 18 pg*hr/ml. In some embodiments,administration of the pessary to a patient provides a correctedgeometric mean area under the curve (AUC)0-24 of estradiol that is lessthan about 112 pg*hr/ml. For example, administration of the pessary to apatient provides a corrected geometric mean area under the curve(AUC)0-24 of estradiol that is less than about 63 pg*hr/ml.

In some embodiments, administration of the pessary to a patient providesa corrected geometric mean peak plasma concentration (C_(max)) ofestrone that is less than about 14 pg*hr/ml. For example, administrationof the pessary to a patient provides a corrected geometric mean peakplasma concentration (C_(max)) of estrone that is less than about 7pg*hr/ml. In some embodiments, administration of the pessary to apatient provides a corrected geometric mean area under the curve(AUC)0-24 of estrone that is less than about 65 pg*hr/ml. For example,administration of the pessary to a patient provides a correctedgeometric mean area under the curve (AUC)0-24 of estrone that is lessthan about 31 pg*hr/ml.

In some embodiments, administration of the pessary to a patient providesa corrected geometric mean peak plasma concentration (C_(max)) ofestrone sulfate that is less than about 613 pg*hr/ml. For example,administration of the pessary to a patient provides a correctedgeometric mean peak plasma concentration (C_(max)) of estrone sulfatethat is less than about 16 pg*hr/ml. In some embodiments, administrationof the pessary to a patient provides a corrected geometric mean areaunder the curve (AUC)0-24 of estrone sulfate that is less than about5291 pg*hr/ml. For example, administration of the pessary to a patientprovides a corrected geometric mean area under the curve (AUC)0-24 ofestrone sulfate that is less than about 84 pg*hr/ml.

In further aspects of this disclosure, capsule disintegration may bedetermined. In some embodiments, delivery vehicle disintegration orabsorption (presence or absence of the delivery vehicle afteradministration) at day 1 of treatment (for example, at 6 hours postfirst treatment) and at day 15 (for example, one day post the lasttreatment on day 14).

Statistical Measurements

According to embodiments, pharmacokinetics of the pharmaceuticalcomposition disclosed herein are measured using statistical analysis.According to embodiments, Analysis of Variance (“ANOVA”) or Analysis ofCoVariance (“ANCOVA”) are used to evaluate differences between a patientreceiving treatment with a pharmaceutical composition comprising anactive pharmaceutical composition (for example, a pharmaceuticalcomposition comprising estradiol) and a patient receiving treatment witha placebo (for example, the same pharmaceutical composition but withoutestradiol) or a reference drug. A person of ordinary skill in the artwill understand how to perform statistical analysis of the datacollected.

EXAMPLES

The following examples are of pharmaceutical compositions, deliveryvehicles, and combinations thereof. Methods of making are alsodisclosed. Data generated using the pharmaceutical compositionsdisclosed herein are also disclosed.

Example 1: Pharmaceutical Composition

In embodiments, estradiol is procured and combined with one or morepharmaceutically acceptable solubilizing agents. The estradiol ispurchased as a pharmaceutical grade ingredient, often as micronizedestradiol, although other forms can also be used. In embodiments, thepharmaceutical composition comprises estradiol in a dosage strength offrom about 1 μg to about 50 μg. In embodiments, the pharmaceuticalcomposition comprises 10 μg of estradiol. In embodiments, thepharmaceutical composition comprises 25 μg of estradiol.

In embodiments, the estradiol is combined with pharmaceuticallyacceptable solubilizing agents, and, optionally, other excipients, toform a pharmaceutical composition. In embodiments, the solubilizingagent is one or more of CAPMUL MCM, MIGLYOL 812, GELUCIRE 39/01,GELUCIRE 43/01, GELUCIRE 50/13, and TEFOSE 63.

GELUCIRE 39/01 and GELUCIRE 43/01 each have an HLB value of 1. GELUCIRE50/13 has an HLB value of 13. TEFOSE 63 has an HLB value of between 9and 10.

Various combinations of pharmaceutically acceptable solubilizing agentswere combined with estradiol and examined as shown in Table 1.

TABLE 1 Capmul MCM (“MCM”), Gelucire 39/01 (“39/01”), Gelucire43/01(“43/01”), Gelucire 50/13(“50/13”), and Tefose (“Tefose 63”)Physical Physical state @ state @ 37° C. Melting Dispersion Vehicle Roomafter ~30 Viscosity Time @ in water # system Ratio Temperature minutes(cps) 37° C. 37° C. 1 MCM:39/01 8:2 Solid Clear liquid 50 @ Start: 6 minSmall oil 37° C. Finish: 12 min drops on top 2 MCM:39/01 7:3 Solid Clearliquid Start: 9 min Finish: 19 min 3 MCM:39/01 6:4 Solid Clear liquidStart: 20 min Finish: 32 min 4 MCM:43/01 8:2 Solid Liquid with solidparticles 5 MCM:43/01 7:3 Solid Liquid with solid particles 6 MCM:50/139:1 Liquid/ Liquid/cloudy 140@ Clear after Uniformly cloudy 25° C. 20min cloudy dispersion 7 MCM:50/13 8:2 Liquid/ Liquid/cloudy 190@Uniformly cloudy 25° C. cloudy dispersion 8 MCM:50/13 7:3 SemisolidSemisolid 9 MCM:TEFOSE 9:1 Semisolid Liquid/cloudy 150@ Start: 1 minUniformly 63 25° C. Finish: 5 min cloudy dispersion 10 MCM:TEFOSE 8:2Semisolid Semisolid 240@ Uniformly 63 25° C. cloudy dispersion 11MCM:TEFOSE 7:3 Semisolid Semisolid 380@ Semisolid Uniformly 63 25° C.after 30 min cloudy at 37° C., dispersion doesn't melt at 41° C. 12MIGLYOL 9:1 Semisolid Semisolid 140@ 2 phases, 812:50/13 25° C. oil ontop 13 MIGLYOL 9:1 Liquid/ Liquid/cloudy 90@ Start: 1 min 2 phases,812:TEFOSE cloudy 25° C. Finish: 5 min oil on top 63

Pharmaceutical compositions in Table 1 that were liquid or semisolid atroom temperature were tested using a Brookfield viscometer (BrookfieldEngineering Laboratories, Middleboro, Mass.) at room temperature.Pharmaceutical compositions appearing in Table 1 that were solid atambient temperature were tested using a Brookfield viscometer at 37° C.

Pharmaceutical compositions appearing in Table 1 that were solid at roomtemperature were assessed at 37° C. to determine their meltingcharacteristics. The viscosity of the gels can be important duringencapsulation of the formulation. For example, in some cases, it isnecessary to warm the formulation prior to filing of the gelatincapsules. In addition, the melting characteristics of the compositioncan have important implications following administration of theformulation into the body. For example, in some embodiments, theformulation will melt at temperatures below about 37° C. PharmaceuticalComposition 11 (Capmul MCM/Tefose 63), for example, did not melt at 37°C. or 41° C.

A dispersion assessment of the pharmaceutical compositions appearing inTable 1 was performed. The dispersion assessment was performed bytransferring 300 mg of each vehicle system in 100 ml of 37° C. water,without agitation, and observing for mixing characteristics. Resultsvaried from formation of oil drops on the top to separation of phases touniform, but cloudy dispersions. Generally speaking, it is believed thatformulations able to readily disperse in aqueous solution will havebetter dispersion characteristics upon administration. It wassurprisingly found, however, as shown below in Examples 7-9, thatformulations that did not readily disperse in aqueous solution (e.g.,Formulation 13) and instead formed two phases upon introduction to theaqueous solution were found to be the most effective when administeredto the human body.

Example 2: Delivery Vehicle

In embodiments, the pharmaceutical composition is delivered in a gelatincapsule delivery vehicle. The gelatin capsule delivery vehiclecomprises, for example, gelatin (e.g., Gelatin, NF (150 Bloom, Type B)),hydrolyzed collagen (e.g., GELITA®, GELITA AG, Eberbach, Germany),glycerin, sorbitol special, or other excipients in proportions that arewell known and understood by persons of ordinary skill in the art.Sorbitol special may be obtained commercially and may tend to act as aplasticizer and humectant.

A variety of delivery vehicles were developed, as show in Table 2, GelsA through F. In Table 2, each delivery vehicle A through F differs inthe proportion of one or more components.

TABLE 2 Gelatin Capsule Delivery Vehicles A B C D E F Ingredient % w/w %w/w % w/w % w/w % w/w % w/w Gelatin, NF (150 Bloom, Type B) 41.0 41.041.0 41.0 43.0 43.0 Glycerin 99.7%, USP 6.0 6.0 6.0 6.0 18.0 18.0Sorbitol Special, USP 15.0 15.0 15.0 15.0 GELITA ® (hydrolyzed collagen)3 3.0 Citric acid 0.1 0.5 1 0.1 Purified Water 35.0 37.9 37.5 37.0 36.038.9 Total 100.0 100.0 100.0 100.0 100.0 100.0 Dissolution gel strips,Avg of 3 48 min 50 min 75 min 70 min (500 ml DH2O, 50 rpm @ 37° C.) (42,45, 58) (50, 51, 50) (76, 75, 74) (70, 71, 70) Dissolution gel strips,Avg of 3 70 min 78 min 82 min (500 ml pH 4 buffer, 50 rpm @ 37° C.)

Each delivery vehicle A through F was prepared at a temperature rangefrom about 45° C. to about 85° C. Each molten delivery vehicle A throughF was cast into a film, dried, and cut into strips. The strips were cutinto uniform pieces weighing about 0.5 g, with about 0.5 mm thickness.Strips were placed into a USP Type 2 dissolution vessel in either wateror pH 4 buffer solution and the time for them to completely dissolve wasrecorded (see TABLE 2). Delivery vehicle A had the fastest dissolutionin both water and pH 4 buffer solution.

Example 3: Pharmaceutical Compositions and Delivery Vehicle

Various combinations of the pharmaceutical compositions from TABLE 1 andfrom TABLE 2 were prepared. The combinations are shown in TABLE 3.

TABLE 3 Batch Size Delivery Trial Pharmaceutical Composition Ratio gVehicle 1 MCM:39/01 8:2 750 A 2 MCM:50/13 8:2 750 A 3 MCM:TEFOSE 63 8:2750 A 4 MCM:TEFOSE 63 8:2 750 B 5 MIGLYOL 812:TEFOSE 63 9:1 750 A

Each aliquot of the pharmaceutical compositions of Table 3 about 300 mgto about 310 mg. Batch size was as listed in TABLE 3. To encapsulate thevehicle system, each 300 mg to about 310 mg pharmaceutical compositionaliquot was encapsulated in about 200 mg of the gelatin capsule deliveryvehicle. Thus, for example, in Trial 1, the pharmaceutical compositiondenoted by MCM:39/01 was encapsulated in gelatin capsule deliveryvehicle A for a total encapsulated weight of about 500 mg to about 510mg. The aliquot size is arbitrary depending on the concentration of theestradiol and the desired gelatin capsule delivery vehicle size.Artisans will readily understand how to adjust the amount of estradiolin the pharmaceutical composition to accommodate a given size ofdelivery vehicle, when the delivery vehicle encapsulates thepharmaceutical composition.

Example 4: Estradiol Solubility

In various experiments, solubilizing agents were tested to determinewhether they were able to solubilize 2 mg of estradiol for a totalpharmaceutical composition weight of 100 mg. The solubilizing agentswere considered suitable if estradiol solubility in the solubilizingagent was greater than or equal to about 20 mg/g. Initial solubility wasmeasured by dissolving micronized estradiol into various solubilizingagents until the estradiol was saturated (the estradiol/solubilizingagent equilibrated for three days), filtering the undissolved estradiol,and analyzing the resulting pharmaceutical composition for estradiolconcentration by HPLC.

TABLE 4 Solubility of Solubilizing Agents (*denotes literaturereference) Ingredient Solubility (mg/g) PEG 400 105* Propylene Glycol 75* Polysorbate 80  36* TRANSCUTOL HP 141  CAPMUL PG8   31.2

Example 5: Pharmaceutical Compositions

The following pharmaceutical compositions are contemplated.

Gel Mass

Ingredient % w/w Qty/Batch (kg) Gelatin 150 Bloom Limed Bone, NF 41.0082.00 Hydrolyzed Gelatin 3.00 6.00 Glycerin 99.7% 6.00 12.00 SorbitolSpecial, NF 15.00 30.00 Opatint White G-18006 1.20 2.40 Opatine RedDG-15001 0.06 0.12 Purified Water, USP 33.74 67.48 Total 100.00 200.00Kg

Pharmaceutical Composition 1: 10 μg Estradiol

Qty/Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydrate 0.0100.003 0.10 g micronized, USP CAPMUL ® MCM, NF (Glyceryl 240.0 79.9972.40 kg Caprylate/Caprate or Medium Chain Mono- and Diglycerides)GELUCIRE ® 50/13 (stearoyl 60.0 20.0 600.0 g polyoxyl-32 glycerides NF)Total 300.0 100.0 3.0 kg

Pharmaceutical Composition 2: 10 μg Estradiol

Qty/Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydrate 0.0100.003 0.10 g micronized, USP MIGLOYL ® 812 (medium 270.0 89.997 2.70 kgchain triglyceride) TEFOSE ® 63 (mixture of 30.0 10.0 300.0 g PEG-6stearate or ethylene glycol palmitostearate or PEG-32 stearate; polyoxyl6 and polyoxyl 32 palmitostearate/glycol stearate) Total 300.0 100.03.00 kg

Pharmaceutical Composition 3: 25 μg Estradiol

Qty/Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydrate0.026* 0.009 0.26 g micronized, USP MIGLOYL ® 812 (medium 270.0 89.9912.70 kg chain triglyceride) TEFOSE ® 63 (mixture of 30.02 10.0 300.0 gPEG-6 stearate or ethylene glycol palmitostearate or PEG-32 stearate;polyoxyl 6 and polyoxyl 32 palmitostearate/glycol stearate) Total 300.0100.0 3.00 kg *1.0 mg estradiol is equivalent to 1.03 mg estradiolhemihydrate

Pharmaceutical Composition 4: 4 μg Estradiol

Qty/Capsule Ingredients (mg) % w/w Qty/Batch Estradiol hemihydrate0.0041* 0.001 0.041 g micronized, USP MIGLOYL ® 812 (medium 269.9989.999 2700.0 g chain triglyceride) TEFOSE ® 63 (mixture of 30.0 10.0300.0 g PEG-6 stearate or ethylene glycol palmitostearate or PEG-32stearate; polyoxyl 6 and polyoxyl 32 palmitostearate/glycol stearate)Total 300.0 100.0 3000.0 g *1.0 mg estradiol is equivalent to 1.03 mgestradiol hemihydrate

Example 6: Process

FIG. 1 illustrates an embodiment of a method making pharmaceuticalcomposition comprising estradiol solubilized in CapmulMCM/Geluciresolubilizing agent encapsulated in a soft gelatin delivery vehicle 100.In operation 102, the CapmulMCM is heated to 40° C.±5° C. Heating may beaccomplished through any suitable means. The heating may be performed inany suitable vessel, such as a stainless steel vessel. Otherpharmaceutical compositions can be made using the same general method bysubstituting various excipients, including the solubilizing agent.

In operation 104, GELUCIRE is mixed with the CapmulMCM to form thefinished solubilizing agent. As used herein, any form of GELUCIRE may beused in operation 104. For example, one or more of GELUCIRE 39/01,GELUCIRE 43/01, GELUCIRE 50/13 may be used in operation 104. Mixing isperformed as would be known to persons of ordinary skill in the art, forexample by impeller, agitator, stirrer, or other like devices used tomix pharmaceutical compositions. Operation 104 may be performed under aninert or relatively inert gas atmosphere, such as nitrogen gas. Mixingmay be performed in any vessels that are known to persons of ordinaryskill in the art, such as a stainless steel vessel or a steel tank.

In operation 106 estradiol is mixed into the solubilizing agent. Inembodiments, the estradiol in micronized when mixed into thesolubilizing agent. In other embodiments, the estradiol added is in anon-micronized form. Mixing may be facilitated by an impeller, agitator,stirrer, or other like devices used to mix pharmaceutical compositions.Operation 106 may be performed under an inert or relatively inert gasatmosphere, such as nitrogen gas.

In embodiments, however, the addition of estradiol may be performedprior to operation 104. In that regard, operations 104 and 106 areinterchangeable with respect to timing or can be performedcontemporaneously with each other.

In operation 110, the gelatin delivery vehicle is prepared. Any of thegelatin delivery vehicles described herein may be used in operation 110.In embodiments, gelatin, hydrolyzed collagen, glyercin, and otherexcipients are combined at a temperature range from about 45° C. toabout 85° C. and prepared as a film. Mixing may occur in a steel tank orother container used for preparing gelatin delivery vehicles. Mixing maybe facilitated by an impellor, agitator, stirrer, or other devices usedto combine the contents of gelatin delivery vehicles. Operation 110 maybe performed under an inert or relatively inert gas atmosphere, such asnitrogen gas. In embodiments, the gelatin delivery vehicle mixture isdegassed prior to being used to encapsulate the pharmaceuticalcomposition.

In operation 112, the gelatin delivery vehicle encapsulates thepharmaceutical composition, according to protocols well known to personsof ordinary skill in the art. In operation 112, a soft gelatin capsuledelivery vehicle is prepared by combining the pharmaceutical compositionmade in operation 106 with the gelatin delivery vehicle made inoperation 110. The gelatin may be wrapped around the material, partiallyor fully encapsulating it or the gelatin can also be injected orotherwise filled with the pharmaceutical composition made in operation106.

In embodiments, operation 112 is completed in a suitable die to providea desired shape. Vaginal soft gel capsules may be prepared in a varietyof geometries. For example, vaginal soft gel capsules may be shaped as atear drop, a cone with frustoconical end, a cylinder, a cylinder withlarger “cap” portion as illustrated in FIG. 2, or other shapes suitablefor insertion into the vagina. The resulting pharmaceutical compositionencapsulated in the soft gelatin delivery vehicle may be inserteddigitally or with an applicator.

Example 7: Study of Estradiol Pharmaceutical Composition on theImprovement of Vulvovaginal Atrophy (VVA)

The objective of this study was designed to evaluate the efficacy andsafety of a pharmaceutical composition comprising 10 μg estradiol (i.e.,Pharmaceutical Composition 2) in treating moderate to severe symptoms ofVVA associated with menopause after 14 days of treatment, and toestimate the effect size and variability of vulvovaginal atrophyendpoints. In addition, the systemic exposure to estradiol from singleand multiple doses of the pharmaceutical composition was investigated.

This study was a phase 1, randomized, double-blind, placebo-controlledtrial to evaluate safety and efficacy of the pharmaceutical compositionin reducing moderate to severe symptoms of vaginal atrophy associatedwith menopause and to investigate the systemic exposure to estradiolfollowing once daily intravaginal administrations of a pharmaceuticalcomposition for 14 days.

Postmenopausal subjects who met the study entry criteria were randomizedto one of two treatment groups (pharmaceutical composition or placebo).During the screening period subjects were asked to self-assess thesymptoms of VVA, including vaginal dryness, vaginal or vulvar irritationor itching, dysuria, vaginal pain associated with sexual activity, andvaginal bleeding associated with sexual activity. Subjects with at leastone self-assessed moderate to severe symptom of VVA identified by thesubject as being most bothersome to her were eligible to participate inthe study.

Clinical evaluations were performed at the following time points:

Screening Period (up to 28 days);

Visit 1—Randomization/Baseline (day 1);

Visit 2—Interim (day 8); and

Visit 3—End of the treatment (day 15).

Eligible subjects were randomized in a 1:1 ratio to receive eitherpharmaceutical composition comprising estradiol 10 μg or a matchingplacebo vaginal softgel capsule, and self-administered their first doseof study medication at the clinical facility under the supervision ofthe study personnel. Serial blood samples for monitoring of estradiollevel were collected at 0.0, 1.0, 3.0, and 6.0 hours relative to firstdose administration on day 1. Subjects remained at the clinical siteuntil completion of the 6-hour blood draw and returned to clinicalfacility for additional single blood draws for measurement of estradiolconcentration on day 8 (before the morning dose) and day 15. Subjectswere provided with enough study medication until the next scheduledvisit and were instructed to self-administer their assigned studytreatment once a day intravaginally at approximately the same time (±1hour) every morning. Each subject was provided with a diary in which shewas required to daily record investigational drug dosing dates andtimes. Subjects returned to clinical facility on day 8 for interim visitand on day 15 for end of treatment assessments and post studyexaminations. Capsule disintegration state was assessed by theinvestigator at day 1 (6 hours post-dose) and day 15.

The study involved a screening period of up to 28 days beforerandomization and treatment period of 14 days. Selection of dosagestrength (estradiol 10 μg) and treatment regimen (once daily for twoweeks) was based on the FDA findings on safety and efficacy of the RLD.

Number of Subjects (Planned and Analyzed)

Up to 50 (25 per treatment group) postmenopausal female subjects 40 to75 years old with symptoms of moderate to severe VVA were randomized. 50subjects were enrolled, 48 subjects completed the study, and 48 subjectswere analyzed.

Diagnosis and Main Criteria for Inclusion

Fifty female subjects were enrolled in the study. Post-menopausal femalesubjects 40 to 75 years of age, with a mean age was 62.3 years wereenrolled. Subjects' mean weight (kg) was 71.2 kg with a range of44.5-100 kg. Subjects' mean height (cm) was 162.6 cm with a range of149.9-175.2 cm, and the mean BMI (kg/m²) was 26.8 kg/m² with a range of19-33 kg/m². Criteria of inclusion in the study included:self-identification of at least one moderate to severe symptom of VVA,for example, vaginal dryness, dysparuenia, vaginal or vulvar irritation,burning, or itching, dysuria, vaginal bleeding associated with sexualactivity, that was identified by the subject as being most bothersome toher; ≤5% superficial cells on vaginal smear cytology; vaginal pH>5.0;and estradiol level ≤50 pg/ml. Subject who were judged as being inotherwise generally good health on the basis of a pre-study physicalexamination, clinical laboratory tests, pelvic examination, andmammography were enrolled.

Estradiol 10 μg or Placebo, Dose, and Mode of Administration

Subjects were randomly assigned (in 1:1 allocation) to self-administerone of the following treatments intravaginally once daily for 14 days:

-   -   Treatment A: The pharmaceutical composition of Example 5        (Pharmaceutical Composition 2: 10 μg estradiol); or    -   Treatment B: Placebo vaginal softgel capsule, containing the        same formulation as Treatment A, except for the 10 μg of        estradiol.

The estradiol formulation was a tear drop shaped light pink soft gelcapsule. Treatment B had the same composition, appearance, and route ofadministration as the Treatment A, but contained no estradiol.

Duration of Treatment

The study involved a screening period of up to 28 days beforerandomization and a treatment period of 14 days.

Criteria for Evaluation

Efficacy Endpoints:

-   -   Change from baseline (screening) to day 15 in the Maturation        Index (percent of parabasal vaginal cells, superficial vaginal        cells, and intermediate vaginal cells) of the vaginal smear.        Data for this endpoint are shown in Tables 6-8.    -   Change from baseline (screening) to day 15 in vaginal pH. Data        for this endpoint are shown in Table 9.    -   Change from baseline (randomization) to day 15 in severity of        the most bothersome symptoms: (1) vaginal dryness; (2) vaginal        or vulvar irritation, burning, or itching; (3) dysuria; (4)        dysparuenia; (5) vaginal bleeding associated with sexual        activity. Data for this endpoint are shown in Tables 13 and 15.    -   Change from baseline (randomization) to day 15 in investigator's        assessment of the vaginal mucosa. Data for this endpoint are        shown in Tables 18-21.

Unless otherwise noted, the efficacy endpoints were measured as achange-from Visit 1—Randomization/Baseline (day 1) to Visit 3—End of thetreatment (day 15), except for vaginal bleeding which was expressed aseither treatment success or failure.

Other endpoints include:

-   -   Vital signs, weight, changes in physical exam, pelvic and breast        exam, and adverse events were evaluated as part of the safety        endpoints.    -   Concentration of estradiol at each sampling time.    -   Peak concentration of estradiol on day 1 and sampling time at        which peak occurred.    -   Delivery vehicle disintegration to measure the amount of        residual delivery vehicle remains in the vagina post treatment.

Results from the assessment of plasma concentrations of estradiol arepresented in Table 5.

TABLE 5 Safety Results: The descriptive statistics for Day 1 plasmaestradiol C_(max) and T_(max) are provided below. Estradiol 10 μgPlacebo C_(max) T_(max) C_(max) T_(max) N 24 24 26 26 Mean ± SD 30.7 ±7.47 2.12 ± 1.73 27.5 ± 17.26 4.00 ± 2.68 Geometric 29.9 — 24.7 — MeanMedian 29.8 1.00 22.1 6.00 Min, Max 19.7, 52.3 1.00, 6.00 15.1, 90.00.00, 6.00 CV % 24.3% 81.3% 62.9% 67.1%

Other Endpoints:

Maturation Index Results

Vaginal cytology data was collected as vaginal smears from the lateralvaginal walls according to standard procedures to evaluate vaginalcytology at screening and Visit 3—End of treatment (day 15). The changein the Maturation Index was assessed as a change in cell compositionmeasured at Visit 1—Baseline (day 1) compared to the cell compositionmeasured at Visit 3—End of treatment (day 15). The change in percentageof superficial, parabasal, and intermediate cells obtained from thevaginal mucosal epithelium from a vaginal smear was recorded. Resultsfrom these assessments are presented in Tables 6, 7, and 8.

TABLE 6 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in the Maturation Index of the Vaginal Smear(Percent Parabasal Cells) Difference Estradiol Between 10 μg vs.Estradiol Treatment 90% CI for Placebo Population Statistics 10 μgPlacebo Means Difference P-value Intent-to- N 24  24 — — — Treat Least-−54.4 −4.80 −49.6 (−60.4, −38.8) <0.0001 Squares Mean Mean ± SD −53.8 ±39.7 −5.4 ± 22.3 — — — Median −60.0 −5.0 — — — Min, Max −100.0, 0.0−60.0, 60.0 — — — ¹ Confidence interval for the estradiol 10 μg-Placebofrom ANCOVA with treatment as a fixed effect and baseline as acovariate. ² P-value for treatment comparison from ANCOVA with treatmentas a fixed effect and baseline as a covariate.

TABLE 7 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in the Maturation Index of the Vaginal Smear(Superficial Cells) Difference Estradiol Between 10 μg vs. EstradiolTreatment 90% CI for Placebo Population Statistics 10 μg Placebo MeansDifference P-value Intent-to- N 24   24 — — — Treat Least- 35.2 8.7526.5 (15.4, 37.6) 0.0002 Squares Mean Mean ± SD 35.2 ± 26.4 8.8 ± 18.7 —— — Median 40.0 0.0 — — — Min, Max 0.0, 80.0 0.0, 90.0 — — — ¹Confidence interval for the estradiol 10 μg-Placebo from ANOVA withtreatment as a fixed effect. ² P-value for treatment comparison fromANOVA with treatment as a fixed effect.

TABLE 8 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in the Maturation Index of the Vaginal Smear(Intermediate Cells) Difference Estradiol Between 10 μg vs. EstradiolTreatment 90% CI for Placebo Population Statistics 10 μg Placebo MeansDifference P-value² Intent-to- N 24   24 — — — Treat Least- 18.7 −3.5422.3 (11.1, 33.5) 0.0017 Squares Mean Mean ± SD 18.5 ± 42.7 −3.3 ± 21.6— — — Median 22.5 −5.0 — — — Min, Max −60.0, 100.0 −60.0, 20.0 — — — ¹Confidence interval for the estradiol 10 μg-Placebo from ANCOVA withtreatment as a fixed effect and baseline as a covariate. ²P-value fortreatment comparison from ANCOVA with treatment as a fixed effect andbaseline as a covariate.

Change in pH Results

Vaginal pH was measured at Screening and Visit 3—End of treatment (day15). The pH measurement was obtained by pressing a pH indicator stripagainst the vaginal wall. The subjects entering the study were requiredto have a vaginal pH value greater than 5.0 at screening. pH values wererecorded on the subject's case report form. The subjects were advisednot to have sexual activity and to refrain from using vaginal douchingwithin 24 hours prior to the measurement. Results from these assessmentsare presented in Table 9.

TABLE 9 Primary Efficacy Analysis Results of Change from Baseline(Screening) to Day 15 in Vaginal pH Difference Estradiol Between 10 μgvs. Estradiol Treatment 90% CI for Placebo Population Statistics 10 μgPlacebo Means Difference¹ P-value² Intent-to- N 24 24 — — — Treat Least-−0.974 −0.339 −0.635 (−0.900, −0.368) 0.0002 Squares Mean Mean ± SD−0.917 ± 0.686 −0.396 ± 0.659 — — — Median −1.00 −0.500 — — — Min, Max−2.00, 0.500 −1.50, 0.500 — — — ¹Confidence interval for the estradiol10 μg-Placebo from ANCOVA with treatment as a fixed effect and baselineas a covariate. ²P-value for treatment comparison from ANCOVA withtreatment as a fixed effect and baseline as a covariate.

Most Bothersome Symptoms Data

Subjects were asked to specify the symptom that she identified as the“most bothersome symptom.” During the screening period all of thesubjects were provided with a questionnaire to self-assess the symptomsof VVA: (1) vaginal dryness; (2) vaginal or vulvar irritation, burning,or itching; (3) dysuria; (4) dysparuenia; (5) vaginal bleedingassociated with sexual activity. Each symptom, with the exception ofvaginal bleeding associated with sexual activity, was measured on ascale of 0 to 3, where 0=none, 1=mild, 2=moderate, and 3=severe. Vaginalbleeding associated with sexual activity was measured in a binary scale:N=no bleeding; Y=bleeding. The subject's responses were recorded. Allrandomized subjects were also provided a questionnaire to self-assessthe symptoms of VVA at Visit 1—Randomization/Baseline (day 1) and atVisit 3—End of the treatment (day 15). Subjects recorded theirself-assessments daily in a diary and answers were collected on days 8and 15 (end of treatment). Pre-dose evaluation results obtained at Visit1 were considered as baseline data for the statistical analyses. Datafrom these assessments are presented in Tables 10 and 11.

TABLE 10 Baseline Characteristics for Vaginal Atrophy Symptoms (ITTPopulation) Estradiol 10 μg Estradiol vs. Placebo WA Symptom Statistics10 μg Placebo P-value¹ Vaginal dryness N of 24 24 — Subjects Mean 2.2922.375 0.68231 Vaginal or vulvar N of 24 24 — irritation/ Subjectsburning/itching Mean 0.875 1.333 0.08721 Pain, burning or N of 24 24 —stinging when Subjects urinating Mean 0.583 0.625 0.87681 Vaginal pain Nof 12 12 — associated Subjects² with sexual Mean 2.083 2.333 0.54281activity Vaginal bleeding N of 12 12 associated with Subjects² sexualactivity Percent³ 25.00 33.33 0.31463 ¹P-value for treatment comparisonfrom ANOVA/ANCOVA with treatment as a fixed effect and Baseline as acovariate when appropriate. ²N = number of subjects sexually active atbaseline. ³Percent of subjects with bleeding, evaluated using Fisher'sExact Test.

TABLE 11 Additional Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Severity of Vaginal Atrophy SymptomsDifference Estradiol Least-Squares Mean Between 10 μg vs. StatisticalEstradiol Treatment 90% CI for Placebo Symptom Method¹ 10 μg PlaceboMeans Difference² P-value Vaginal ANCOVA 0.980 0.729 0.251 −0.706,0.204) 0.3597 dryness Vaginal or ANCOVA 0.694 0.514 0.180 −0.549, 0.189)0.4159 vulvar Irritation/ buring/ itching Pain/Burning/ ANCOVA 0.3910.359 0.032 −0.263, 0.200) 0.8185 Stinging (Urination) Vaginal painANOVA 0.800 0.500 0.300 −1.033, 0.433) 0.4872 associated with sexualactivity ¹ANOVA model contained a fixed effect for treatment. ANCOVAadded baseline as a covariate to the model. ²Confidence interval for thedifference between estradiol 10 μg and Placebo treatment least-squaresmeans.

Changes to the most bothersome symptom from the baseline was scoredaccording to the evaluation of VVA symptoms generally set forth above.Tables 13 and 14 show a comparison between the pharmaceuticalcomposition 1 and placebo generally for most bothersome symptom andvaginal atrophy symptom. It is noteworthy to point out that thesemeasurement demonstrated a trend of improvement, though notstatistically significant, at day 15.

TABLE 13 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Severity of the Most Bothersome VVADifference Estradiol Between 10 μg vs. Estradiol Treatment 90% CI forPlacebo Population Statistics 10 μg Placebo Means Difference¹ P-value²Intent-to- N 24 24 — — — Treat Least- −1.043 −1.042 −0.002 (−0.497,0.493) 0.9951 Squares Mean Mean ± SD −1.043 ± 0.928 −1.042 ± 1.08 — — —Median −1.00 −1.00 — — — Min, Max −3.00, 0.00 −3.00, 0.00 — — —¹Confidence interval for the estradiol 10 μg-Placebo from ANOVA withtreatment as a fixed effect. ²P-value for treatment comparison fromANOVA with treatment as a fixed effect.

TABLE 14 Additional Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Severity of Vaginal Atrophy SymptomsSymptom TX-12- Least-Squares Difference 004-HR Mean Between vs.Statistical TX-12- Treatment 90% CI for Placebo Symptom Method¹ 004-HRPlacebo Means Difference² P-value Dryness ANCOVA −0.980 −0.729 −0.251(−0.706, 0.204) 0.3597 Irritation ANCOVA −0.694 −0.514 −0.180 (−0.549,0.189) 0.4159 Pain (Sex) ANOVA −0.800 −0.500 −0.300 (−1.033, 0.433)0.4872 Pain/Burning/ ANCOVA −0.391 −0.359 −0.032 (−0.263, 0.200) 0.8185Stinging (Urination) ¹ANOVA model contained a fixed effect fortreatment. ANCOVA added baseline as a covariate to the model.²Confidence interval for the difference between TX-12-004-HR and Placebotreatment least-squares means.

With respect to the most bothersome symptoms data presented in Tables 13and 14, the period over which the data was measured is generallyconsidered insufficient to make meaningful conclusions. However, thetrends observed as part of this study suggest that the data will showimprovement of the most bothersome symptoms when data for a longer timeperiod is collected.

The absence or presence of any vaginal bleeding associated with sexualactivity was also measured as one of the most bothersome symptoms. Thedata for vaginal bleeding associated with sexual activity is reported inTable 15.

TABLE 15 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Vaginal Bleeding Associated with SexualActivity Baseline (Randomization) and Day 15 Summary of Vaginal BleedingBleeding/ Bleeding/ No Bleeding/ No Bleeding/ No Bleeding BleedingBleeding No Bleeding Treatment N* (Success)² (Failure) (Failure) (NC)Estradiol 10 2 (100%) 0 0 8 10 μg Placebo 10 1 (20%) 3 1 5 P-Value for0.1429 — — — Estradiol 10 μg vs. Placebo¹ *N = Total number of patientswithin each treatment group who were sexually active at both Baselineand Day 15 and provided a response at both visits. NC = No Change - notconsidered in the statistical comparison. ¹P-value for treatmentcomparison from Fisher's Exact Test. ²Percent is based on the number ofsubjects classified as either a Success or a Failure (N = 2 forestradiol 10 μg; N = 5 for Placebo

Estradiol Level/Pharmacokinetics Data

In this study, the systemic exposure to estradiol following once dailyintravaginal administration of estradiol 10 μg for 14 days wasinvestigated. Descriptive statistics of the plasma estradiolconcentrations taken at each sampling time and the observed C_(max) andT_(max) values were recorded in Tables 16 and 17. No statisticallysignificant difference in the systemic concentration of estradiol 10 μgversus the placebo group was observed, which suggests the estradiol isnot carried into the blood stream where it will have a systemic effect.Rather, it remains in localized tissues; the effect of estradiol istherefore believed be local to the location of administration (i.e., thevagina). The lower limits of detection of the assays used to measure thepharmacokinetic data may have affected the measured the accuracy of thepk values presented. Additional pk studies were performed with moreaccurate assays in Examples 8 and 9.

For the purpose of monitoring the estradiol level during the study bloodsamples were collected at 0.0, 1.0, 3.0, and 6.0 hours relative todosing on day 1; prior to dosing on day 8; and prior to dosing on day15. Efforts were made to collect blood samples at their scheduled times.Sample collection and handling procedures for measurement of estradiolblood level was performed according to procedure approved by the sponsorand principal investigator. All baseline and post-treatment plasmaestradiol concentrations were determined using a validated bioanalytical(UPLC-MS/MS) methods. These data are shown in Tables 16 and 17.

TABLE 16 Descriptive Statistics of Estradiol Concentrations (pg/ml) atEach Sampling Time Sampling Time Pre-dose Pre-dose Treatment 0 Hour 1Hour 3 Hours 6 Hours Day 8 Day 15 Estradiol 10 μg N 24   24   24   24  24   22   Mean ± SD 20.1 ± 5.74 28.7 ± 5.89 25.7 ± 5.71 23.4 ± 7.91 21.4 ± 9.28  23.4 ± 8.72  Median 20.2 28.9 24.7 22.3 20.7 20.7 Min, Max2.63, 38.3 18.8, 43.9 19.3, 47.5 3.31, 52.3 2.09, 52.2 17.9, 54.7Placebo N 26   26   26   26   25   24   Mean ± SD 20.5 ± 4.29 21.0 ±6.14 19.0 ± 5.92 26.9 ± 17.36 29.9 ± 22.51 28.1 ± 16.80 Median 20.8 20.820.9 21.7 21.6 21.1 Min, Max 4.03, 29.1 3.19, 41.2 3.15, 26.9 15.1, 90.015.0, 116.2 14.7, 81.3

TABLE 17 Descriptive Statistics of Estradiol C_(max) and T_(max) on Day1 Estradiol 10 μg Placebo C_(max) T_(max) C_(max) T_(max) N 24 24 26 26Mean ± SD 30.7 ± 7.47 2.12 ± 1.73 27.5 ± 17.26 4.00 ± 2.68 Geometric29.9 — 24.7 — Mean Median 29.8 1.00 22.1 6.00 Min, Max 19.7, 52.3 1.00,6.00 15.1, 90.0 0.00, 6.00 CV % 24.3% 81.3% 62.9% 67.1%

Assessment of Vaginal Mucosa Data

The investigators rated the vaginal mucosal appearance at day 1(pre-dose) and day 15. Vaginal color, vaginal epithelial integrity,vaginal epithelial surface thickness, and vaginal secretions wereevaluated according to the following degrees of severity: none, mild,moderate, or severe using scales 0 to 3, where 0=none, 1=mild,2=moderate, and 3=severe. Results from these investigators ratedassessments are presented in Tables 18, 19, 20, and 21.

TABLE 18 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Color) Difference Estradiol Between 10 μgvs. Estradiol Treatment 90% CI for Placebo Population Statistics 10 μgPlacebo Means Difference¹ P-value² Intent-to- N 24 24 — — — Treat Least-−0.199 −0.009 −0.191 (−0.434, 0.052) 0.1945 squares Mean Mean ± SD−0.333 ± 0.565 0.125 ± 0.741 — — — Median 0.00 0.00 — — — Min, Max−2.00, 0.00 −1.00, 2.00 — — — ¹Confidence interval for the estradiol 10μg-Placebo from ANCOVA with treatment as a fixed effect and baseline asa covariate. ²P-value for treatment comparison from ANCOVA withtreatment as a fixed effect and baseline as a covariate.

TABLE 19 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Epithelial Integrity) Difference EstradiolBetween 10 μg vs. Estradiol Treatment 90% CI for Placebo PopulationStatistics 10 μg Placebo Means Difference¹ P-value² Intent-to- N 24 24 —— — Treat Least- −0.342 0.176 −0.518 (−0.726, −0.311) 0.0001 squaresMean Mean ± SD −0.417 ± 0.584 0.250 ± 0.442 — — — Median 0.00 0.00 — — —Min, Max −1.00, 1.00 0.00, 1.00 — — — ¹Confidence interval for theestradiol 10 μg-Placebo from ANCOVA with treatment as a fixed effect andbaseline as a covariate. ²P-value for treatment comparison from ANCOVAwith treatment as a fixed effect and baseline as a covariate.

TABLE 20 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Epithelial Surface Thickness) DifferenceEstradiol Between 10 μg vs. Estradiol Treatment 90% CI for PlaceboPopulation Statistics 10 μg Placebo Means Difference¹ P-value²Intent-to- N 24 24 — — — Treat Least- −0.034 −0.133 0.099 (−0.024,0.221) 0.1820 squares Mean Mean ± SD −0.125 ± 0.338 −0.042 ± 0.550 — — —Median 0.00 0.00 — — — Min, Max −1.00, 0.00 −1.00, 1.00 — — —¹Confidence interval for the estradiol 10 μg-Placebo from ANCOVA withtreatment as a fixed effect and baseline as a covariate. ²P-value fortreatment comparison from ANCOVA with treatment as a fixed effect andbaseline as a covariate.

TABLE 21 Primary Efficacy Analysis Results of Change from Baseline(Randomization) to Day 15 in Investigator's Assessment of the VaginalMucosa (Assessment of Vaginal Secretions) Difference Estradiol Between10 μg vs. Estradiol Treatment 90% CI for Placebo Population Statistics10 μg Placebo Means Difference¹ P-value² Intent-to- N 24 24 — — — TreatLeast- −0.643 −0.274 −0.369 (−0.661, −0.076) 0.0401 squares Mean Mean ±SD −0.792 ± 0.779 −0.125 ± 0.741 — — — Median −1.00 0.00 — — — Min, Max−2.00, 1.00 −2.00, 2.00 — — — ¹Confidence interval for the estradiol 10μg-Placebo from ANCOVA with treatment as a fixed effect and baseline asa covariate. ²P-value for treatment comparison from ANCOVA withtreatment as a fixed effect and baseline as a covariate.

Delivery Vehicle Disintegration Data

Assessment of capsule disintegration in the vagina (presence or absence)at Day 1 (6 hours after dosing) and Day 15. Results of this assessmentis presented in Table 22.

TABLE 22 Capsule Disintegration State in the Vagina on Day 1 and Day 15Estradiol 10 μg Placebo Day 1 Day 15 Day 1 Day 15 No evidence 23 (95.8%)24 (100.0%) 26 (100.0%) 24 (92.3%) of capsule present Evidence 0 (0.0%)0 (0.0%) 0 (0.0%) 0 (0.0%) of capsule present Assessment 1 (4.2%) 0(0.0%) 0 (0.0%) 22 (7.7%) not done

Serum hormone level data was collected to measure the serumconcentrations of estradiol. These data were used for screeninginclusion and were determined using standard clinical chemistry methods.

Appropriateness of Measurements

The selection of the efficacy measurements used in this study was basedon FDA's recommendations for studies of estrogen and estrogen/progestindrug products for the treatment of moderate to severe vasomotor symptomsassociated with the menopause and moderate to severe symptoms of vulvarand vaginal atrophy associated with the menopause (Food and DrugAdministration, Guidance for Industry, Estrogen and Estrogen/ProgestinDrug Products to Treat Vasomotor Symptoms and Vulvar and Vaginal AtrophySymptoms—Recommendations for Clinical Evaluation. January 2003, herebyincorporated by reference).

Standard clinical, laboratory, and statistical procedures were utilizedin the trial. All clinical laboratory procedures were generally acceptedand met quality standards.

Statistical Methods:

Efficacy:

Analysis of variance (ANOVA) was used to evaluate the change frombaseline differences between the subjects receiving estradiol 10 μg andplacebo capsules for all efficacy endpoints, except for vaginalbleeding, to estimate the effect size and variability of the effect. Insome cases, for example, for some vaginal atrophy symptoms, the changefrom baseline (post dose response) was correlated with the baselinevalue (p<0.05), so baseline was included as a covariate to adjust forthis correlation (Analysis of Covariance, ANCOVA). The 90% confidenceintervals on the differences between estradiol 10 μg and placeboendpoint means were determined to evaluate the effect size. The changefrom baseline in vaginal bleeding associated with sexual activity wasevaluated in terms of the proportion of subjects who had treatmentsuccess or failure. Any subject reporting bleeding at baseline who didnot report bleeding at Day 15 was considered to have been successfullytreated. Any subject reporting bleeding at day 15 was considered atreatment failure, regardless of whether they reported baseline bleedingor not. Subjects reporting no bleeding at both baseline and day 15 wereclassified as no-change and were excluded from the statisticalevaluation. The difference in the proportion of subjects with successbetween the two treatment groups was statistically evaluated usingFisher's Exact Test. Results of this difference in proportion arepresented in Table 10.

Measurements of Treatment Compliance

Subjects were required to complete a diary in order to record treatmentcompliance. Diaries were reviewed for treatment compliance at day 8 andday 15 visits. A total of 45 subjects (21 subjects in the estradiol 10μg group and 24 subjects in the placebo group) were 10% compliant withthe treatment regimen.

Due to the investigative nature of the study, no adjustments were madefor multiplicity of endpoints.

Safety:

The frequency and severity of all adverse events were summarizeddescriptively by treatment group.

Results: All forty eight (48) subjects who completed the study wereincluded in the primary efficacy analyses. The results of efficacyanalyses are presented throughout Tables 5, 6, and 7.

CONCLUSIONS

Efficacy

The two-week treatment with pharmaceutical composition 10 μg led to astatistically significant greater mean decrease in percent of parabasalcells than did placebo treatment (54% vs. 5%, p<0.0001), as illustratedin Table 6. At the same time, a significantly greater mean increase inthe percent of superficial cells was observed with the pharmaceuticalcomposition (35%) than with the placebo capsules (9%), with thedifference being highly statistically significant (p=0.0002), asillustrated in Table 7. The difference in pH reduction between thepharmaceutical composition (0.97 units) compared to that for the placebo(0.34 units) was only slightly greater than 0.5 units, but thedifference was detected as statistically significant (p=0.0002), asillustrated in Table 9.

While the decrease in severity of the most bothersome symptom wasessentially the same (˜1 unit) for both pharmaceutical composition andplacebo, the reductions in the severity of the individual symptoms ofvaginal dryness, irritation and pain during sexual activity were allmarginally better for the active treatment than for the placebotreatment. None of the differences between the two treatments, all ofwhich were ≤0.3 units, were detected as statistically significant. Therewas no difference between the two treatments in regard to reduction ofpain/burning/stinging during urination (˜0.4 unit reduction). The lengthof the study was not long enough to show a separation between the mostbothersome symptoms in the pharmaceutical composition and placebo.However, the trends of most bothersome symptoms suggest that with asuitable period of time, significantly significant differences betweenthe two treatments would be observed.

The two-week treatment with estradiol 10 μg capsules showed nostatistically detectable difference in regard to reduction of severityfrom baseline according to the investigator's assessment of vaginalcolor or vaginal epithelial surface thickness. Pharmaceuticalcomposition capsules did demonstrate a statistically significant greaterreduction than did placebo in severity of atrophic effects on vaginalepithelial integrity (−0.34 vs. 0.18, p=0.0001) and vaginal secretions(−0.64 vs. −0.27, p=0.0401).

Descriptive statistical analyses (mean, median, geometric mean, standarddeviation, CV, minimum and maximum, C_(max), and T_(max)) were conductedon the estradiol concentrations at each sampling time, the peakconcentration on day 1 and the time of peak concentration. Results fromthis assessment are presented in Tables 16 and 17.

A pharmaceutical composition comprising estradiol 10 μg outperformedplacebo treatment in regard to improvement in the Maturation Index,reduction in vaginal pH, reduction in the atrophic effects on epithelialintegrity and vaginal secretions. The lack of statistical significancebetween the two treatments in regard to reduction of severity for themost bothersome symptom, and the individual vaginal atrophy symptoms ofdryness, irritation, pain associated with sexual activity, andpain/burning/stinging during urination, is not unexpected given thesmall number of subjects in the study and the short duration of therapy.Too few subjects in the study had vaginal bleeding associated withsexual activity to permit any meaningful evaluation of this vaginalatrophy symptom.

Of the 48 subjects enrolled in the study, 45 subjects were 100%compliant with the treatment regimen. Of the remaining three subjects,one removed herself from the study due to personal reasons and the othertwo subjects each missed one dose due to an adverse event.

Safety

Although the Day 1 mean plasma estradiol peak concentration for thepharmaceutical composition was somewhat higher than that for the Placebo(ratio of geometric means=1.21:Test Product (estradiol 10 μg)21%>Placebo), no statistically significant difference was determined.However, the assay methods were questionable, resulting in questionablepk data. Additional pk studies were performed in Examples 8 and 9.

There were no serious adverse events in the study.

Overall, the pharmaceutical composition comprising estradiol 10 μg waswell tolerated when administered intravaginally in once daily regimenfor 14 days.

Example 8: pk Study (25 μg Formulation)

A pk study was undertaken to compare the 25 μg formulation disclosedherein (Pharmaceutical Composition 3) to the RLD. The results of the pkstudy for estradiol are summarized in Table 23. The p values for thesedata demonstrate statistical significance, as shown in Table 24.

TABLE 23 Statistical Summary of the Comparative Bioavailability Data forUnscaled Average BE studies of Estradiol, Least Square Geometric Meansof Estradiol, Ratio of Means and 90% Confidence Intervals, Fasting/FedBioequivalence Study (Study No.: ESTR-1K-500-12); Dose 25 μg estradiolRatio Parameter Test N RLD N (%) 90% C.I. C_(max) 23.0839 36 42.7024 3654.06 44.18-66.14 (pg/mL) AUC₀₋₂₄ 89.2093 36 292.0606 36 30.5423.72-39.34 (pg · hr/mL)

TABLE 24 P-values for table 23 P-Value Effect C_(max) AUC₀₋₂₄ Treatment<.0001 <.0001 Sequence 0.4478 0.5124 Period 0.4104 0.7221

As illustrated in Table 23, baseline adjusted pk data illustrates thatthe formulations disclosed herein unexpectedly show a 54% decrease inC_(max) and a 31% decrease in the AUC relative to the RLD. This resultis desirable because the estradiol is intended only for localabsorption. These data suggest a decrease in the circulating levels ofestradiol relative to the RLD. Moreover, it is noteworthy to point outthat the C_(max) and AUC levels of estradiol relative to placebo are notstatistically differentiable, which suggests that the formulationsdisclosed herein have a negligible systemic effect. As shown in Table24, there was no significant difference between the test and referenceproducts due to sequence and period effects. However, there was asignificant difference due to treatment effect for both C_(max) and AUC.

Pharmacokinetics for circulating total estrone, a metabolite ofestradiol, is show in Table 25. These data show that the totalcirculating estrone for the formulations disclosed herein resulted in a55% decrease in the C_(max) for circulating estrone, and a 70% decreasein the AUC for circulating estrone.

TABLE 26 P-values for table 25 P-Value Effect C_(max) AUC₀₋₂₄ Treatment0.0002 <.0001 Sequence 0.1524 0.0464 Period 0.0719 0.0118

TABLE 25 Statistical Summary of the Comparative Bioavailability Data forUnscaled Average BE studies of Estrone, Least Square Geometric Means,Ratio of Means and 90% Confidence Intervals, Fasting/Fed BioequivalenceStudy (Study No.: ESTR-1K-500-12); Dose 25 μg estradiol Ratio ParameterTest N RLD N (%) 90% C.I. C_(max) 10.7928 36 23.5794 36 45.77 32.95 to63.59 (pg/mL) AUC₀₋₂₄ 51.2491 36 165.4664 36 30.97 19.8-48.45 (pg ·hr/mL)

There was a significant difference between test and reference productsdue to treatment effect whereas there was no significant difference dueto sequence and period effects for C_(max). For AUC, there was asignificant difference between test and reference products due totreatment, sequence, and period effects.

pk for circulating total estrone sulfate is shown in Table 27. Thesedata show that the total circulating estrone sulfate for thepharmaceutical compositions disclosed herein resulted in a 33% decreasein the C_(max) and a 42% decrease in the AUC for circulating estronesulfate.

TABLE 27 Statistical Summary of the Comparative Bioavailability Data forUnsealed Average BE studies of Estrone Sulfate, Least Square GeometricMeans of Estrone Sulfate, Ratio of Means and 90% Confidence Intervals,Fasting/Fed Bioequivalence Study (Study No.: ESTR-1K-500-12); Dose 25 μgestradiol Ratio Parameter Test N RLD N (%) 90% C.I. C_(max) 490.0449 36730.5605 36 67.08 53.84-83.57 (pg/mL) AUC₀₋₂₄ 4232.9914 36 7323.0827 3657.80 43.23-77.29 (pg · hr/mL)

TABLE 28 P-values for table 27 P-Value Effect C_(max) AUC₀₋₂₄ Treatment0.0042 0.0031 Sequence 0.5035 0.9091 Period 0.1879 0.8804

There was a significant difference between test and reference productsdue to treatment effect whereas there was no significant difference duesequence and period effects for both C_(max) and AUC.

Example 9: pk Study (10 μg Formulation)

A pk study was undertaken to compare the 10 μg formulation disclosedherein (Pharmaceutical Composition 2) to the RLD. The results of the pkstudy for estradiol are summarized in Table 29-40, and FIGS. 9-14.

A pk study was undertaken to compare pharmaceutical compositionsdisclosed herein having 10 μg of estradiol to the RLD. The results ofthe pk study for estradiol are summarized in tables 29-34, whichdemonstrate that the pharmaceutical compositions disclosed herein moreeffectively prevented systemic absorption of the estradiol. Table 35shows that the pharmaceutical compositions disclosed herein had a 28%improvement over the RLD for systemic blood concentration C_(max) and72% AUC improvement over the RLD.

TABLE 29 Summary of Pharmacokinetic Parameters of Test product (T) ofEstradiol - Baseline adjusted (N = 34) Arithmetic Pharmacokinetic Mean ±Standard Coefficient Parameter Deviation of Variation Median MinimumMaximum C_(max) (pg/mL) 15.7176 ± 7.9179  50.3761 13.9000 6.5000 49.6000AUC₀₋₂₄ 53.0100 ± 19.5629 36.9041 49.9750 24.3000 95.1500 (pg · hr/mL)t_(max) (hr) 1.98 ± 1.29 65.34 2.00 1.00 8.05

TABLE 30 Summary of Pharmacokinetic Parameters of Reference product (R)of Estradiol - Baseline adjusted (N = 34) Arithmetic PharmacokineticMean ± Standard Coefficient Parameter Deviation of Variation MedianMinimum Maximum C_(max) (pg/mL)  24.1882 ± 11.9218 49.2877 24.15001.0000 55.3000 AUC₀₋₂₄ 163.8586 ± 72.0913 43.9960 158.0375 2.0000304.8500 (pg · hr/mL) t_(max) (hr) 10.53 ± 5.58 52.94 8.06 2.00 24.00

TABLE 31 Geometric Mean of Test Product (T) and Reference product (R) ofEstradiol - Baseline adjusted (N = 34) Geometric Mean PharmacokineticParameter Test Product (T) Reference Product (R) C_(max) (pg/mL) 14.377420.3837 AUC₀₋₂₄ (pg · hr/mL) 49.6231 132.9218 t_(max) (hr) 1.75 9.28

TABLE 32 Statistical Results of Test product (T) versus Referenceproduct (R) for Estradiol - Baseline adjusted (N = 34) Geometric LeastSquare Mean Test Reference Intra T/R 90% Pharmacokinetic Product ProductSubject Ratio Confidence Parameter (T) (R) CV % % Interval C_(max)(pg/mL) 14.4490 20.1980 60.68 71.54* 56.82-90.08 AUC₀₋₂₄ 49.7310131.0400 70.64 37.95* 29.21-49.31 (pg · hr/mL) *Comparison was detectedas statistically significant by ANOVA (α = 0.05).

The pk data for total estrone likewise demonstrated reduced systemicexposure when compared to the RLD. Table 33 shows the pharmaceuticalcompositions disclosed herein reduced systemic exposure by 25% forC_(max) and 49% for AUC.

TABLE 33 Summary of Pharmacokinetic Parameters of Test product (T) ofEstrone - Baseline adjusted (N = 33) Arithmetic Pharmacokinetic Mean ±Standard Coefficient Parameter Deviation of Variation Median MinimumMaximum C_(max) (pg/mL) 6.8485 ± 6.5824 96.1149 5.4000 1.3000 36.3000AUC₀₋₂₄ 34.7051 ± 27.9541 80.5476 30.8500 3.3500 116.7500 (pg · hr/mL)t_(max) (hr) 9.12 ± 8.83 96.80 4.00 1.00 24.00

TABLE 34 Summary of Pharmacokinetic Parameters of Reference product (R)of Estrone - Baseline adjusted (N = 33) Arithmetic Pharmacokinetic Mean± Standard Coefficient Parameter Deviation of Variation Median MinimumMaximum C_(max) (pg/mL) 8.8333 ± 7.1469 80.9086 6.7000 2.7000 30.3000AUC₀₋₂₄ 63.0042 ± 46.5484 73.8814 51.2800 8.8000 214.0000 (pg · hr/mL)t_(max) (hr) 11.16 ± 7.24  64.95 10.00 4.00 24.00

TABLE 35 Geometric Mean of Test Product (T) and Reference product (R) ofEstrone - Baseline adjusted (N = 33) Geometric Mean PharmacokineticParameter Test Product (T) Reference Product (R) C_(max) (pg/mL) 5.15076.9773 AUC₀₋₂₄ (pg · hr/mL) 24.2426 48.2377 t_(max) (hr) 5.87 9.07

TABLE 36 Statistical Results of Test product (T) versus Referenceproduct (R) for Estrone - Baseline adjusted (N = 33) Geometric LeastSquare Mean Test Reference Intra T/R 90% Pharmacokinetic Product ProductSubject Ratio Confidence Parameter (T) (R) CV % % Interval C_(max)(pg/mL) 5.1620 6.9280 47.59 74.50* 61.69-89.97 AUC₀₋₂₄ 24.1960 47.902073.66 50.51* 38.37-66.50 (pg · hr/mL) *Comparison was detected asstatistically significant by ANOVA (α = 0.05).

The pk data for estrone sulfate likewise demonstrated reduced systemicexposure when compared to the RLD. Table 37 shows the pharmaceuticalcompositions disclosed herein reduced systemic exposure by 25% forC_(max) and 42% for AUC.

TABLE 37 Summary of Pharmacokinetic Parameters of Test product (T) ofEstrone Sulfate - Baseline adjusted (N = 24) Arithmetic PharmacokineticMean ± Standard Coefficient Parameter Deviation of Variation MedianMinimum Maximum C_(max) (ng/mL) 13.9042 ± 7.0402  50.6339 11.1500 1.300039.0000 AUC₀₋₂₄ 97.9953 ± 80.8861 82.5408 76.2750 5.1025 338.0000 (ng ·hr/mL) t_(max) (hr) 6.33 ± 4.56 71.93 4.00 4.00 24.00

TABLE 38 Summary of Pharmacokinetic Parameters of Reference product (R)of Estrone Sulfate - Baseline adjusted (N = 24) ArithmeticPharmacokinetic Mean ± Standard Coefficient Parameter Deviation ofVariation Median Minimum Maximum C_(max) (ng/mL) 19.2542 ± 11.363359.0173 15.2000 7.0000 53.7000 AUC₀₋₂₄ 177.6208 ± 166.2408 93.5931124.0000 20.0000 683.0500 (ng · hr/mL) t_(max) (hr) 10.33 ± 5.58 54.0510.00 2.00 24.00

TABLE 39 Geometric Mean of Test Product (T) and Reference product (R) ofEstrone Sulfate - Baseline adjusted (N = 24) Geometric MeanPharmacokinetic Parameter Test Product (T) Reference Product (R) C_(max)(ng/mL) 12.1579 16.8587 AUC₀₋₂₄ (ng · hr/mL) 66.5996 121.5597 t_(max)(hr) 5.49 8.83

TABLE 40 Statistical Results of Test product (T) versus Referenceproduct (R) for Estrone Sulfate - Baseline adjusted (N = 24) GeometricLeast Square Mean Test Reference Intra T/R 90% Pharmacokinetic ProductProduct Subject Ratio Confidence Parameter (T) (R) CV % % IntervalC_(max) (ng/mL) 12.3350 16.5470 48.02 74.55* 59.43-93.51 AUC₀₋₂₄ 68.5260118.4170 73.87 57.87* 41.68-80.35 (ng · hr/mL) *Comparison was detectedas statistically significant by ANOVA (α = 0.05).

While the pharmaceutical compositions and methods have been described interms of what are presently considered to be practical and preferredembodiments, it is to be understood that the disclosure need not belimited to the disclosed embodiments. It is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the claims, the scope of which should be accorded the broadestinterpretation so as to encompass all such modifications and similarembodiments. This disclosure includes any and all embodiments of thefollowing claims.

What is claimed is:
 1. A method for treating vulvovaginal atrophy in ahuman subject comprising: intravaginally administering estradiol to thesubject by manually inserting about two inches into the vagina a liquidpharmaceutical composition comprising about 4 μg to about 25 μg ofestradiol, about 90 wt % of a mixture of caprylic and caprictriglyceride, and about 10 wt % of a mixture of PEG-6 stearate, ethyleneglycol palmitostearate and PEG-32 stearate, wherein the composition hasa viscosity from about 50 cP to about 1000 cP at 25° C., whereinestradiol is the only active hormone in the composition, wherein thecomposition comprises all of the estradiol and is encapsulated in acapsule, and wherein the composition is inserted while the subject is ina reclining position or in a standing position; wherein administrationof the composition results in a decrease in the vulvovaginal atrophy inthe subject within two to six weeks from the first administration. 2.The method of claim 1, wherein the capsule is a soft gelatin capsule. 3.The method of claim 1, wherein administration of the composition resultsin an increase in the percentage of vaginal superficial cells within twoweeks from the first administration.
 4. The method of claim 1, whereinadministration of the composition results in a decrease in thepercentage of vaginal parabasal cells within two weeks from the firstadministration.
 5. The method of claim 1, wherein administration of thecomposition results in a decrease in vaginal pH within two weeks fromthe first administration.
 6. The method of claim 1, wherein theadministration comprises inserting the composition once daily for twoweeks and twice weekly thereafter.
 7. The method of claim 1, wherein thevulvovaginal atrophy is due to menopause.
 8. The method of claim 1,wherein the composition contains 4 μg estradiol or 10 μg estradiol. 9.The method of claim 1, wherein the viscosity of the composition is fromabout 50 cP to about 380 cP at 25° C.